Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Incidence, immunological and clinical characteristics of reactivation of latent Toxoplasma gondii infection in HIV-infected patients.

To determine changes in incidence of reactivation of Toxoplasma gondii infection, manifesting as toxoplasmic encephalitis, and to assess the immunological mechanisms controlling reactivation in HIV-infected patients, a Czech cohort of 502 HIV/T. gondii co-infected patients was followed for 2909·3 person-years. The incidence of toxoplasmic encephalitis between the periods before and after the introduction of combination antiretroviral therapy (cART) was compared. Toxoplasmic encephalitis was diagnosed in 21 patients. In those patients the geometric mean value of CD4+ T lymphocytes was 12·6 times lower than in patients with non-reactivated T. gondii infection but an additionally significant decline in CD8+ T lymphocytes (3·3-fold) and natural killer cells (4·3-fold) was observed. This confirms the significance of these parameters. A twelvefold decrease in Toxoplasma reactivation incidence (40·2 vs. 3·4/1000 person-years) between monitored periods was seen. In the cART era, Toxoplasma reactivation was observed only in patients with unrecognized HIV infection or refusing therapy.

[Toxoplasmosis in immunocompromised patients]. / Toxoplasmóza u imunokompromitovaných pacientu.

In humans, toxoplasmosis mostly occurs as a latent infection, but in immunocompromised individuals, the agent may reactivate and cause severe to life-threatening disease. HIV positive individuals and transplant recipients, in particular hematopoietic stem cell transplant and heart transplant recipients, are at highest risk. The disease most often affects the central nervous system but can involve any organ. Because of the alteration of the immune response in these patients, the serodiagnosis is not reliable and direct detection of the causative agent is needed--namely by microscopy and DNA PCR. If inadequately treated or left untreated, toxoplasmosis generally has a fatal prognosis in immunocompromised patients and therefore, the treatment must be started as early and energetically as possible. The gold standard both in the treatment of reactivation and secondary prophylaxis is the pyrimethamine-sulfadiazine combination while co-trimoxazole can be used in the primary prophylaxis for high-risk patients.

Differences in onset of disease and severity of psychopathology between toxoplasmosis-related and toxoplasmosis-unrelated schizophrenia.

OBJECTIVE: Toxoplasmosis is a lifelong parasitic disease that appears to be associated to schizophrenia. However, no distinguishing attributes in Toxoplasma-infected schizophrenia patients have been described as yet. METHOD: We searched for differences in symptom profile, cognitive performance and treatment response between 194 Toxoplasma-free and 57 (22.7%) Toxoplasma-infected schizophrenia patients treated in Prague Psychiatric Centre between 2000 and 2010. RESULTS: Infected and non-infected patients differed in severity of symptoms (P = 0.032) measured with the Positive and Negative Symptom Scale (PANSS). Infected patients scored higher in positive subscale of PANSS, but not in the general and negative subscales. Infected men scored higher also in Total PANSS score, and negative, reality distortion, disorganisation and cognitive scores. Higher PANSS scores of positive, negative and disorganised psychopathology were associated with the lower titres of anti-Toxoplasma antibodies suggesting that psychopathology deteriorates with duration of parasitic infection. Infected patients remained about 33 days longer in hospital during their last admission than uninfected ones (P = 0.003). Schizophrenia started approximately 1 year earlier in infected men and about 3 years later in infected women, no such difference was observed in uninfected subjects. CONCLUSION: Latent toxoplasmosis in schizophrenia may lead to more severe positive psychopathology and perhaps less favourable course of schizophrenia.

Antibody response of HIV-infected patients to latent, cerebral and recently acquired toxoplasmosis.

The aim of this longitudinal study with 626 HIV-infected patients was to evaluate the capability of serological tests in diagnosing the presence of Toxoplasma gondii infection in HIV-infected patients, as well as the potential impact of various treatment regimes on serological results. Low IgG antibody levels and stable or declining titres predominated. IgM positivity occurred in ten patients (one seroconversion, seven latent, two cerebral toxoplasmosis). Complement fixation test (CFT) titres >or=1:32 imply that the relative risk of cerebral toxoplasmosis is 6.84 (95% confidence interval [CI] 1.44-32.5) but with a predictive value of only 14.0% (95% CI 5.3-27.9). Values of specific antibodies are not biassed by antiretroviral treatment and/or prophylaxis for toxoplasmosis, and the detection of specific antibodies is very useful in the identification of T. gondii infection in the HIV-infected population, but the role of serology in predicting the clinical manifestation of T. gondii infection is limited.

Sex-dependent toxoplasmosis-associated differences in testosterone concentration in humans.

Several lines of indirect evidence suggest that subjects with latent infection of the coccidian parasite Toxoplasma gondii have a higher concentration of testosterone than uninfected controls. Here, we searched for direct evidence of latent toxoplasmosis-associated differences in testosterone concentration among a population of 174 female and 91 male students screened for Toxoplasma infection. We have found Toxoplasma-infected men to have a higher concentration of testosterone and Toxoplasma-infected women to have a lower concentration of testosterone than Toxoplasma-free controls. The opposite direction of the testosterone shift in men compared to women can explain the observed gender specificity of behavioural shifts in Toxoplasma-infected subjects.

Evaluation of a commercial IgE ELISA in comparison with IgA and IgM ELISAs, IgG avidity assay and complement fixation for the diagnosis of acute toxoplasmosis.

A panel of sera from patients with known case histories representative of acute toxoplasmosis (primarily lymphadenopathy, n = 106), latent toxoplasmosis (asymptomatic, n = 368) and negative samples (n = 54) was used to evaluate the capacity of five serological tests to differentiate among patients with acute or latent toxoplasmosis and non-infected individuals. Positive IgA, IgE and IgM ELISA results and low IgG avidity and complement fixation test (CFT) titres of >or=256 were considered to be indicative of acute toxoplasmosis. The most sensitive methods were IgM ELISA (98.1%) and CFT (97.1%), albeit with low specificity (65.0% and 64.5%, respectively) and positive predictive values (43.3% and 42.7%, respectively). IgG avidity assay and IgE ELISA had the highest specificity (97.7% and 91.7%, respectively) and the highest positive predictive values (89.4% and 75.6%, respectively). The best association between serological results and clinical findings was obtained with IgE ELISA (86%, as expressed via Youden's index). In a subset of 259 samples categorised by the period between the onset of clinical symptoms and sampling, >50% of patients had enlarged lymph nodes for <4 months, despite a broad range of differences. However, IgM remained positive for 12-18 months, IgA for 6-9 months and IgE for 4-6 months. IgG avidity remained low for a maximum of 4 months, after which avidity increased despite the persistence of enlarged lymph nodes and a positive IgE assay. Detection of IgE appears to be a highly specific test for confirming the acute nature of Toxoplasma infections that have been detected by other sensitive methods.

New and old risk-factors for Toxoplasma gondii infection: prospective cross-sectional study among military personnel in the Czech Republic.

The aims of this study were to evaluate seroprevalence and the importance of various risk-factors for Toxoplasma infection in the Czech Republic. A prospective cross-sectional survey was conducted among military personnel in Prague. Consenting subjects (n = 3250) completed a questionnaire concerning demographics and risk-factors, and blood samples were taken to determine anti-Toxoplasma antibody titres according to complement fixation and ELISA IgG and IgM tests. The seroprevalence of toxoplasmosis was 23%. In multivariate analysis, independent predictors of Toxoplasma seropositivity were age (OR 1.03 / year), consumption of raw meat (OR 1.35), owning a cat (OR 1.25), owning rabbits (OR 1.47), childhood residence in a town with a population of <10 000 inhabitants (OR 1.63) vs. location of the childhood residence in a town with population of >100 000 inhabitants, and blood group type A (OR 1.28), B (OR 1.33) or AB (OR 1.43) vs. O. These results suggested that horizontal toxoplasmosis transmission in the Czech Republic may occur through consumption of raw meat, contact with cat faeces and farming.

[Recommendation for diagnostics and therapy of toxoplasmosis in persons with HIV infection]. / Doporucený postup diagnostiky a terapie toxoplazmózy u osob s HIV infekcí.

Reactivation of latent toxoplasmosis is a serious complication in patients with deep immunodeficiency, but the disease has a good prognosis if early diagnosed and effectively treated. Definitive etiologic proof of the reactivation may be difficult and thus an empiric method (therapeutic trial) is used for confirmation of the diagnosis in clinical practice. The preferred therapy is a combination of pyrimethamine + sulfadiazine.

Correlation of duration of latent Toxoplasma gondii infection with personality changes in women.

Many parasites induce characteristic changes in behavior of their hosts. In humans latent toxoplasmosis is associated with changes in personality profiles. It has been already shown that a decrease in superego strength is correlated with duration of toxoplasmosis in men. Here we studied changes in personality profiles with Cattell's 16 PF questionnaire in Toxoplasma-infected women. The changes were measured as differences in personality factors between Toxoplasma-infected subjects and uninfected controls of the same age. The low-rate changes were studied in 230 women diagnosed with acute toxoplasmosis during past 14 years. The results showed the correlation between duration of toxoplasmosis and level of factors G (high superego strength) and Q3 (high strength of self sentiment). The high-rate changes were estimated by measuring the correlation between level of Toxoplasma-antibody titers (which rapidly decline after the end of acute phase of toxoplasmosis) and personality factors in an experimental set of 55 young mothers with latent toxoplasmosis. Again, certain factors, namely A (affectothymia), F (surgence), G (high superego strength), H (parmia), and L (protension), correlated with the length of the infection. We suggest that the parasite induced the changes in the personality profiles of the women because of our observation of an increasingly different personality profile over time between women with latent infection and controls. The same evidence questions the view that women with a particular personality profile are more prone to acquisition of T. gondii infection.

Modulation of murine lymphocyte responsiveness by the saliva of Rhodnius prolixus (Hemiptera: Reduviidae).

Incorporation of 3H-thymidine by splenic lymphocytes was studied in CBA mice that were bitten by the triatomine bug Rhodnius prolixus (Stål) 14, 7, 4, and 2 d before lymphocyte isolation, respectively. In bitten mice, both spontaneous and mitogen-induced proliferative responses were reduced. The most pronounced effect was observed 4 d after exposure when lymphocytes were significantly suppressed in proliferative response to the mitogens concanavalin A, phytohemagglutinin, and bacterial lipopolysaccharide. The maximum inhibition caused an 85% reduction of proliferation and was observed in phytohemagglutinin-stimulated cell cultures. The immunosuppressive effect was caused neither by stress nor by an nonspecific cytotoxic effect of R. prolixus saliva. The described immunosuppressive activity of saliva could aid in successful repeated feedings of R. prolixus on the same host and possibly could play a role in transmission of Trypanosoma cruzi.

The effect of drugs on the mortality of mice inoculated with Friend leukaemia virus or toxoplasma gondii.

Infection and tumors provoke substantial changes accompanied with the disbalance of many neuroendocrine factors which in their summarizing effects influence the life span of animals. Our previous results showed enhanced mortality after one injection of morphine in association with Friend leukaemia virus infection. The aim of this study was to examine the effects of some other opioids (pethidine and pentazocine) and an acetylcholine esterase inhibitor neostigmine on the survival of animals under two conditions: (1) Friend leukaemia virus infection which mostly depressed immune functions, and (2) Toxoplasma gondii infection which in general enhanced the immune status. In contrast to our previous observation with morphine, the mortality induced by single doses of pethidine (150 mg/kg) or pentazocine (50-75 mg/kg) was unchanged during the Friend leukaemia virus infection. A single injection of neostigmine (0.42 or 0.56 mg/kg) was significantly more lethal in DBA-2 mice infected with Friend leukaemia virus. Neostigmine in doses of 0.33 and 0.4 mg/kg caused death in 46 % and 57 %, respectively, of animals infected with Toxoplasma gondii which was significantly higher in comparison with only 8 % and 12.5 % in control groups. Pethidine (150 mg/kg) killed 70 % of Toxoplasma gondii infected animals and even 90 % of non-infected mice. Thus, the Friend leukaemia virus and Toxoplasma gondii infections increased toxicity only of some drugs which may, at least partly, be associated with altered immune status during infection and involvement of the cholinergic system.

Transient nature of Toxoplasma gondii-induced behavioral changes in mice.

Many parasites induce specific changes in host behavior that promote the transmission of their infective stages between hosts. Toxoplasmosis in rodents is known to be accompanied by specific behavioral changes (shift in activity level, learning capacity, and novelty discrimination) that can theoretically increase the chance of infected animals being eaten by the definitive host, the cat. However, toxoplasmosis is also accompanied by many pathological symptoms. It is not known whether the behavioral changes are products of manipulation activity of the parasite or only nonspecific by-products of pathological symptoms of toxoplasmosis. Here, we compared the dynamics of development of behavioral and pathological changes in Toxoplasma gondii-infected mice. The results showed that the maximum reduction of mouse activity corresponded with the peak of pathological symptoms, and also that maximum increase of reaction times corresponded with the peak of development of tissue cysts in the brains of infected mice. Behavioral changes were only transient and disappeared before the 12th wk postinoculation. The results suggest that the behavioral changes in infected mice reported by many authors and observed in our experiments could be nonspecific by-products of pathological symptoms of toxoplasmosis rather than specific products of manipulation activity by the parasite.

[Diagnosis of acquired toxoplasmosis using simultaneous determination of specific immunoglobulins M, A and E]. / Diagnostika získané toxoplazmózy pomocí simultánního stanovení specifických imunoglobulinu trídy M, A a E.

Simultaneous estimation of specific immunoglobulins M,A,E was tested in sera of 57 patients with confirmed acquired toxoplasmosis (24 acute and 33 subacute or chronic) using immunosorbent agglutination assay (ISAGA) and immunofluorescent tests (toxoplasma antibody-total and IgM). The antigen for ISAGA was prepared from tissue cultures HeLa infected with Toxoplasma gondii virulent strain P. Toxoplasmas were maintained in tissue cultures continuously for 18 years. High levels of IgM antibodies were found in all 24 patients during the acute phase of toxoplasmosis (0-5 month after onset). IgA antibodies were found also in all but one and IgE in 71% of these patients. IgM antibodies persisted for the longest period and it was possible to find them in 14% of patients 3 years or longer after the onset, IgA antibodies persisted for 9 months similarly as IgE (with one exception after 2 years). Simultaneous estimation of specific immunoglobulins M,A,E combined with immunofluorescent tests makes the diagnosis of Toxoplasma infection more reliable.

Parasitic opportunistic infections in Czech HIV-infected patients--a prospective study.

To determine the frequency of opportunistic parasites, especially causative agents of microsporidiosis and cryptosporidiosis in HIV-infected patients in the Czech Republic, more than 75% of all Czech HIV-infected patients were examined during this study. Target staining techniques were used for parasitological examination of stool, sputum and urine of HIV-infected patients. In addition, their sera were examined by indirect ELISA technique with specific antigens from E. cuniculi, E. hellem and C. parvum. Specific antibodies to T. gondii were detected by CF test. In 2.1% of HIV-infected patients microsporidia E. bieneusi and E. intestinalis were found by parasitological examination, 5.3% of HIV-infected patients were seropositive to E. cuniculi and 1.3% to E. intestinalis were found by parasitological examination, 5.3% of HIV-infected patients were seropositive to E. cuniculi and 1.3% to E. hellem antigens in the high titre of 600. All blood donors (control group) were seronegative in these titres. C. parvum oocysts were found in one AIDS patient, 10.5% patients were seropositive to C. parvum in higher titres (600, 1800). Occurrence of T. gondii antibodies was not significantly greater in HIV-seropositive patients (27.5%) than in the control group (21.4%). P. carinii was detected in 13.6% of the patients. This study shows the quite low occurrence of opportunistic parasitoses among Czech HIV-infected patients. Although the high prevalence of specific antibodies against causative agents of microsporidiosis and cryptosporidiosis was recorded, the number of clinical cases was low.

Plesiomonas shigelloides in water and fish.

Plesiomonas shigelloides strains isolated from aquariums, surface water and fish were serotyped using the international antigenic scheme and a provisional scheme designed for environmental strains, called the "Schubert" scheme. A remarkable variety of serovars represented in both schemes was found, namely in surface water samples and in fish.

Poorer results of mice with latent toxoplasmosis in learning tests: impaired learning processes or the novelty discrimination mechanism?

The heteroxenous protozoan parasite Toxoplasma gondii is transmitted from the intermediate host (any warm-blooded animal) to the definitive host (members of the felidae) by carnivory. The infected intermediate hosts develop several specific behavioural changes that are usually considered products of manipulative activity of the parasite aimed to increase the probability of its transmission to the definitive host. Among other changes, the infected rodents were shown to have impaired learning capability. All previous studies were done 2-6 weeks after the infection. Therefore, it was difficult to resolve whether the observed impairment of learning processes was a result of acute or latent toxoplasmosis, i.e. whether it was a side-effect of the disease or a product of manipulation activity. Here we studied the learning capability of Toxoplasma-infected mice in the static rod test and 8-arm radial maze test and their spontaneous activity in the wheel running test 10 weeks after the infection. The infected mice achieved worse scores in the learning tests but showed higher spontaneous activity in the wheel running test. However, a detailed study of the obtained results as well as of the data reported by other authors suggested that the differences between infected and control mice were a result of impaired ability to recognize novel stimuli rather than of impaired learning capacity in animals with latent toxoplasmosis.

Influence of latent toxoplasmosis on the secondary sex ratio in mice.

The sex ratio may be influenced by many factors, such as stress and immunosuppression, age of parents, parity and sex of preceding siblings. In animal systems, parasitism often changes the sex ratio of infected hosts, which can increase the probability of their transmission. The most common human protozoan parasite in developed countries, Toxoplasma gondii (prevalence 20%-80%), is known to change the behaviour of its intermediate hosts, thereby increasing the probability of transmission to its definitive host (the cat) by predation. The intermediate hosts, which under natural conditions are rodents, serve as the vector for Toxoplasma. Therefore, we speculate that Toxoplasma can alter the secondary sex ratio (i.e. male to female ratio in the offspring) of infected females to increase the proportion of (congenitally infected) male offspring, which are the more migratory sex in most rodent species. Here we studied the sex ratio of experimentally infected laboratory mice, expressed here as the proportion of males in the litter. In accordance with our hypothesis and results of previous retrospective cohort studies on human subjects, mice with toxoplasmosis produced a higher sex ratio than controls, in the early phase of latent infection. In the later phase of infection, mice with congenital toxoplasmosis had a lower sex ratio than controls, which is in accord with the Trivers-Willard hypothesis of sex ratio manipulation, suggesting that females in poor physical condition give birth to more female offspring.

The role of dopamine in Toxoplasma-induced behavioural alterations in mice: an ethological and ethopharmacological study.

Toxoplasma gondii, a cosmopolitan protozoan parasite, is known to induce behavioural alterations in rodents and may exert an effect on human personality and behaviour. The mechanism of parasite-induced alterations in host behaviour has not been described, but it was hypothesized that development of Toxoplasma tissue cysts in the brain could affect the dopaminergic neuromodulatory system. In this study, we tested the effect of latent Toxoplasma infection on mouse behaviour associated with activity of the dopaminergic system, i.e. locomotion in a novel environment and exploration test. Additionally, we examined the behavioural response of Toxoplasma-infected mice to a selective dopamine uptake inhibitor, GBR 12909. In both genders, Toxoplasma infection decreased locomotion in the open field. Infected females displayed an increased level of exploration in the holeboard test. GBR 12909 induced suppression in holeboard-exploration in the infected males, but had an opposite effect on the controls. These results suggest an association between Toxoplasma gondii infection and changes in the dopaminergic neuromodulatory system.

Induction of changes in human behaviour by the parasitic protozoan Toxoplasma gondii.

Toxoplasma gondii, the coccidian parasite, is known to induce changes in the behaviour of its intermediate hosts. The high prevalence of this parasite in the human population (20-80%) offers the opportunity of studying the influence of the parasite on human behaviour by screening of a normal population. A total of 224 men and 170 women were tested for toxoplasmosis and their personality profiles were measured by Cattell's questionnaire. Highly significant differences between Toxoplasma-infected and uninfected subjects were observed (P < 0.01). For men the factors G (low superego strength), L (protension), O (guilt proneness), and Q2 (group dependency) were positively influenced in infected subjects. For women the prevailing factors were A (affectothymia, P < 0.01), L (alaxia), O (untroubled adequacy) and Q2 (self-sufficiency). To reveal whether toxoplasmosis induces personality factor-shifts or whether certain combinations of personality factors influence the probability of acquiring Toxoplasma infection, we examined the personality profiles of 164 male patients diagnosed with acute toxoplasmosis during the past 13 years. The existence of a positive correlation between the duration of latent toxoplasmosis and the intensity of superego strength decrease (P < 0.02) suggested that the decrease of superego strength (the willingness to accept group moral standards) was induced by T. gondii infection.