Seroepidemiology of Toxoplasma gondii Infection among Healthy Blood Donors in Taiwan

Toxoplasma gondii is an opportunistic, zoonotic pathogen with a worldwide distribution. There are large variations in the seroprevalence of T. gondii infection in different regions of the world. Although toxoplasmosis became a notifiable communicable disease in Taiwan in 2007, little is known about its epidemiology among the general population. This cross-sectional study aimed to survey the seroprevalence of T. gondii infection and its risk factors among healthy blood donors in Taiwan. Through collaborating with the Taiwan Blood Services Foundation, a total of 1,783 healthy blood donors from all six-branch blood service centers participated in this study. The blood samples were tested for the presence of T. gondii antibodies and DNA using enzyme immunoassays and real-time PCR, respectively. Structured questionnaires were used to gather information on risk factors for T. gondii infection. Of the 1,783 participants, 166 (9.3%) tested positive for anti-Toxoplasma IgG, while 5 (0.28%) tested positive for anti-Toxoplasma IgM. The five IgM positive donors had high avidity antibodies suggestive of past infection. No active parasitemia was detected by real-time PCR assays. Multivariate logistic regression showed that undercooked pork meat consumption (adjusted odds ratio [OR] = 2.9; 95% confidence interval [CI]: 1.3–6.5), raw mussels consumption (adjusted OR = 5.3; 95% CI: 1.5–19.1), having a cat in the household (adjusted OR = 2.0; 95% CI: 1.2–3.2), a lower education level (adjusted OR = 1.6; 95% CI: 1.1–2.3), and donation place in eastern Taiwan (adjusted OR = 2.5; 95% CI: 1.6–3.9) were independent risk factors for Toxoplasma seropositivity. These findings provide information on the seroprevalence and epidemiology of T. gondii infection among healthy blood donors in Taiwan.     

The involvement of heparin in retinal infection by Toxoplasma gondii in a chick model revealed an ontogenetic-dependent pattern

This work aimed to test the influence of heparin on the susceptibility of retinal cells to Toxoplasma gondii infection. Primary cultures of retinas from chick embryos of 8 (E8) or 11 (E11) days and fibroblasts (control) were used. To determine the influence of heparin in T. gondii infection, tachyzoites of the RH strain were treated with heparin before addition in the culture. A monoclonal anti-heparin antibody was used to analyze the heparin distribution on fibroblast and retinal cell surfaces. Our results showed that retinal cells (E8 and E11) had a higher infection rate than fibroblasts (91% and 24% versus 13%, respectively). Pre-treatment of T. gondii with heparin decreased infection of E8 retinal cells when compared with non-treated parasites (45% versus 91%, respectively), but not of E11 cells (35% versus 48%). In accordance, retinal cells presented an intense heparin staining by immunofluorescence assay. In conclusion, retinal cells from chick embryos were more susceptible to infection by T. gondii compared to fibroblasts and, pre-treatment of tachyzoites with heparin decreased the number of infected cells and parasite burden particularly for E8 retinal cells.     

Using Molecular Epidemiology to Track Toxoplasma gondii from Terrestrial Carnivores to Marine Hosts: Implications for Public Health and Conservation

Background

Environmental transmission of the zoonotic parasite Toxoplasma gondii, which is shed only by felids, poses risks to human and animal health in temperate and tropical ecosystems. Atypical T. gondii genotypes have been linked to severe disease in people and the threatened population of California sea otters. To investigate land-to-sea parasite transmission, we screened 373 carnivores (feral domestic cats, mountain lions, bobcats, foxes, and coyotes) for T. gondii infection and examined the distribution of genotypes in 85 infected animals sampled near the sea otter range.

Methodology/Principal Findings

Nested PCR-RFLP analyses and direct DNA sequencing at six independent polymorphic genetic loci (B1, SAG1, SAG3, GRA6, L358, and Apico) were used to characterize T. gondii strains in infected animals. Strains consistent with Type X, a novel genotype previously identified in over 70% of infected sea otters and four terrestrial wild carnivores along the California coast, were detected in all sampled species, including domestic cats. However, odds of Type X infection were 14 times higher (95% CI: 1.3–148.6) for wild felids than feral domestic cats. Type X infection was also linked to undeveloped lands (OR = 22, 95% CI: 2.3–250.7). A spatial cluster of terrestrial Type II infection (P = 0.04) was identified in developed lands bordering an area of increased risk for sea otter Type II infection. Two spatial clusters of animals infected with strains consistent with Type X (P≤0.01) were detected in less developed landscapes.

Conclusions

Differences in T. gondii genotype prevalence among domestic and wild felids, as well as the spatial distribution of genotypes, suggest co-existing domestic and wild T. gondii transmission cycles that likely overlap at the interface of developed and undeveloped lands. Anthropogenic development driving contact between these cycles may increase atypical T. gondii genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife.     

Auranofin Is Highly Efficacious against Toxoplasma gondii In Vitro and in an In Vivo Experimental Model of Acute Toxoplasmosis

Background

The mainstay of toxoplasmosis treatment targets the folate biosynthetic pathways and has not changed for the last 50 years. The activity of these chemotherapeutic agents is restricted to one lifecycle stage of Toxoplasma gondii, they have significant toxicity, and the impending threat of emerging resistance to these agents makes the discovery of new therapies a priority. We now demonstrate that auranofin, an orally administered gold containing compound that was FDA approved for treatment of rheumatoid arthritis, has activity against Toxoplasma gondii in vitro (IC₅₀ = 0.28 µM) and in vivo (1 mg/kg).

Methods/Principal Findings

Replication within human foreskin fibroblasts of RH tachyzoites was inhibited by auranofin. At 0.4 µM, auranofin inhibited replication, as measured by percent infected fibroblasts at 24 hrs, (10.94% vs. 24.66% of controls; p = 0.0003) with no effect on parasite invasion (16.95% vs. 12.91% p = 0.4331). After 18 hrs, 62% of extracellular parasites treated with auranofin were non-viable compared to control using an ATP viability assay (p = 0.0003). In vivo, a previously standardized chicken embryo model of acute toxoplasmosis was used. Fourteen day old chicken embryos were injected through the chorioallantoic vein with 1×10⁴ tachyzoites of the virulent RH strain. The treatment group received one dose of auranofin at the time of inoculation (1 mg/kg estimated body weight). On day 5, auranofin-treated chicken embryos were 100% protected against death (p = 0.0002) and had a significantly reduced parasite load as determined by histopathology, immunohistochemistry and by the number of parasites quantified by real-time PCR.

Conclusions

These results reveal in vitro and in vivo activity of auranofin against T. gondii, suggesting that it may be an effective alternative treatment for toxoplasmosis.     

Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptide-microarray

Background

Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals.

Methodology

A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100).

Findings

The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II–III, type I–III or type I–II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers.

Conclusions

Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution.     

Exposure to Multiple Parasites Is Associated with the Prevalence of Active Convulsive Epilepsy in Sub-Saharan Africa

Background

Epilepsy is common in developing countries, and it is often associated with parasitic infections. We investigated the relationship between exposure to parasitic infections, particularly multiple infections and active convulsive epilepsy (ACE), in five sites across sub-Saharan Africa.

Methods and Findings

A case-control design that matched on age and location was used. Blood samples were collected from 986 prevalent cases and 1,313 age-matched community controls and tested for presence of antibodies to Onchocerca volvulus, Toxocara canis, Toxoplasma gondii, Plasmodium falciparum, Taenia solium and HIV. Exposure (seropositivity) to Onchocerca volvulus (OR = 1.98; 95%CI: 1.52–2.58, p<0.001), Toxocara canis (OR = 1.52; 95%CI: 1.23–1.87, p<0.001), Toxoplasma gondii (OR = 1.28; 95%CI: 1.04–1.56, p = 0.018) and higher antibody levels (top tertile) to Toxocara canis (OR = 1.70; 95%CI: 1.30–2.24, p<0.001) were associated with an increased prevalence of ACE. Exposure to multiple infections was common (73.8% of cases and 65.5% of controls had been exposed to two or more infections), and for T. gondii and O. volvulus co-infection, their combined effect on the prevalence of ACE, as determined by the relative excess risk due to interaction (RERI), was more than additive (T. gondii and O. volvulus, RERI = 1.19). The prevalence of T. solium antibodies was low (2.8% of cases and 2.2% of controls) and was not associated with ACE in the study areas.

Conclusion

This study investigates how the degree of exposure to parasites and multiple parasitic infections are associated with ACE and may explain conflicting results obtained when only seropositivity is considered. The findings from this study should be further validated.     

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.     

Toxoplasma gondii Protein Disulfide Isomerase (TgPDI) Is a Novel Vaccine Candidate against Toxoplasmosis

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect all warm-blooded animals, including both mammals and birds. Protein disulfide isomerase (PDI) localises to the surface of T. gondii tachyzoites and modulates the interactions between parasite and host cells. In this study, the protective efficacy of recombinant T. gondii PDI (rTgPDI) as a vaccine candidate against T. gondii infection in BALB/c mice was evaluated. rTgPDI was expressed and purified from Escherichia coli. Five groups of animals (10 animals/group) were immunised with 10, 20, 30, 40 μg of rTgPDI per mouse or with PBS as a control group. All immunisations were performed via the nasal route at 1, 14 and 21 days. Two weeks after the last immunisation, the immune responses were evaluated by lymphoproliferative assays and by cytokine and antibody measurements. The immunised mice were challenged with tachyzoites of the virulent T. gondii RH strain on the 14th day after the last immunisation. Following the challenge, the tachyzoite loads in tissues were assessed, and animal survival time was recorded. Our results showed that the group immunised with 30 μg rTgPDI showed significantly higher levels of specific antibodies against the recombinant protein, a strong lymphoproliferative response and significantly higher levels of IgG2a, IFN-gamma (IFN-γ), IL-2 and IL-4 production compared with other doses and control groups. While no changes in IL-10 levels were detected. After being challenged with T. gondii tachyzoites, the numbers of tachyzoites in brain and liver tissues from the rTgPDI group were significantly reduced compared with those of the control group, and the survival time of the mice in the rTgPDI group was longer than that of mice in the control group. Our results showed that immunisation with rTgPDI elicited a protective immune reaction and suggested that rTgPDI might represent a promising vaccine candidate for combating toxoplasmosis.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

Evaluation of the anti-Toxoplasma gondii Activity of Hederagenin in vitro and in vivo

Toxoplasma gondii infection is widespread worldwide, not only posing a serious threat to human food safety and animal husbandry, but also endangering human health. The selectivity index was employed to measure anti-T. gondii activity. Hederagenin (HE) exhibited potent anti-T. gondii activity and low cytotoxicity. For this reason, HE was selected for in vivo experiments. HE showed 64.8%±13.1% inhibition for peritoneal tachyzoites in mice, higher than spiramycin 56.8%±6.0%. Biochemical parameters such as alanine aminotransferase, aspartate aminotransferase, glutathione, and malondialdehyde, illustrated that HE was a good inhibitor of T. gondii in vivo. This compound was also effective in relieving T. gondii-induced liver damage. Collectively, it was demonstrated that HE had potential as an anti-T. gondii agent.     

Comparison of Toxoplasma gondii Seroprevalence in Shelter Cats and Dogs during 1999–2001 and 2009–2011 in Tokyo,Japan

Toxoplasma gondii is an important human health concern with respect to abortion, congenital hydrocephalus, and encephalitis in immunocompromised people. Cats and dogs both are potential sources of T. gondii because they have close contact with humans. However, no epidemiological surveys have been conducted in Tokyo over the past decade. Therefore, the present study investigated and compared the seroprevalence of T. gondii infection in shelter cats and dogs during 1999–2001 and 2009–2011 in Tokyo, Japan. Serum samples were collected from 337 shelter cats and 325 shelter dogs in urban and suburban areas of Tokyo, during 1999–2001 (233 cats and 219 dogs) and 2009–2011 (104 cats and 106 dogs). T. gondii antibodies were measured in the serum samples using a commercial latex agglutination test. Data were compared using the Fisher’s exact test, and significance was indicated at P < 0.05. The overall seroprevalence of T. gondii infection in cats was 5.6% (13 of 233) in 1999–2001 and 6.7% (7 of 104) in 2009–2011, and that in dogs was 1.8% (4 of 219) and 1.9% (2 of 106), respectively. Significantly higher seroprevalence was observed in cats from suburban areas compared with cats in urban areas during both periods (P < 0.05). These results reveal that there has been little change in the feline and canine seroprevalence over the past decade, indicating that the risk of T. gondii exposure for cats and dogs in Tokyo is considerably low as the seroprevalence has reached a steady state.     

Toxoplasma gondii Chitinase Induces Macrophage Activation

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50°C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.     

Increasing Newly Diagnosed Rate and Changing Risk Factors of HCV in Yanbian Prefecture,a High Endemic Area in China

Background

The newly diagnosed rate of HCV infection is increasing in China. However, the risk factors have not been fully identified. Here, a survey was performed in Yanbian Prefecture, a high-endemic area in China.

Methods

We identified newly diagnosed HCV infection in 2007–2011, using the local National Disease Supervision Information Management System from the Chinese Center for Disease Control and Prevention. We determined the risk factors using a case-control survey by questionnaire.

Results

Yanbian Prefecture had a rapid increase in the yearly newly diagnosed rate of HCV infection from 32.6 to 72.1/100.000 from the year 2007 to 2011. People aged 50–64 years had a high HCV infection of 43.4%, but only 0.3% of cases were reported in those aged less than 20 years. Cosmetic treatment, family history, blood transfusion, and dental treatment were independent risk factors for HCV infection. Unexpectedly, cosmetic treatments [odd ratio (OR) = 5.15, 95% confidence interval (CI) = 2.31–11.48, P = 0.00] and family history (OR = 4.68, 95% CI = 2.67–8.75, P = 0.00) showed a higher risk than the conventional risk factors of blood transfusion (OR = 4.49, 95% CI = 1.95–10.37, P = 0.001) and dental treatment (OR = 2.98, 95% CI = 1.42–6.25, P = 0.00). To further analyze the intrafamilial transmission, we found that spouses of HCV patients had an increased risk for acquiring HCV (OR = 5.75, 95% CI: 1.94–17.07), without significant association between either HCV RNA viral load (P = 0.29) or genotype (P = 0.43).

Conclusions

HCV infection was increased in Yanbian Prefecture. Cosmetic treatment was a higher risk factor than medical procedure. HCV infection had a clear family clustering phenomenon, especially between spouses.     

Genetic Characterization of Toxoplasma gondii Isolates and Toxoplasmosis Seroprevalence in Stray Cats of ?zmir,Turkey

Currently, some Toxoplasma gondii genotypes are being associated with serious clinical presentations. A recent report showing the Africa 1 genotype in two local congenital toxoplasmosis cases acquired in Turkey formed the basis of this study because atypical Africa 1 genotype is most frequently detected in animals and patients from sub-Saharan Africa. Since stray cats are considered as the linkage between wild life and urban life in T. gondii transmission, the present study aimed to isolate and characterize T. gondii strains circulating in stray cats of İzmir (Western Turkey). A secondary objective was to determine toxoplasmosis seroprevalence in this cat population. Tissues obtained from 100 deceased stray cats were bioassayed and isolated strains were genotyped using 15 microsatellite markers. In addition, toxoplasmosis seroprevalence was analyzed in 1121 cat sera collected from several large veterinary clinics in İzmir. Among the 22 isolates, 19 were Type II (86.3%), two were Type III (9%) and one was Africa 1 genotype (4.5%). The overall seropositivity rates in cats were 42–48% and 33.4–34.4% according to IFA and ELISA, respectively. Seroprevalence in deceased cats was significantly higher than in healthy cats (P = 0.0033). Finding both the major clonal Type II lineage together with the Type III lineage also found in Middle East, and an atypical genotype, Africa 1 appears consistent with the specific geographic location of Turkey between three continents and raises the possibility of transportation of these strains between continents through trade routes or long distance migratory birds. In addition, the first large study of toxoplasma seroprevalence in a stray cat population was also reported. The relatively high seropositivity rates and the variety of T. gondii genotypes confirm the local stray cat population as a risk factor for human toxoplasmosis in İzmir.     

Toxocara Seroprevalence among Clinically Healthy Individuals,Pregnant Women and Psychiatric Patients and Associated Risk Factors in Shandong Province,Eastern China

Background

Toxocarosis is a widespread zoonosis caused by the ascarid nematodes Toxocara canis and Toxocara cati, which primarily infect dogs and cats, respectively. Most human infections with Toxocara are asymptomatic; however, some infected individuals may develop a serious illness and even death. Nevertheless, epidemiological knowledge regarding the prevalence and risks associated with Toxocara infection is limited in China. Therefore, we performed a cross-sectional pilot study and estimated the seroprevalence of Toxocara infection in humans in Shandong Province, eastern China for the first time, from June 2011 to July 2013, involving clinically healthy individuals, pregnant women and psychiatric patients, aiming to attract public attention to Toxocara infection.

Methodology/Principle Findings

Seroprevalence of Toxocara was determined using an enzyme-linked immunosorbent assay based on a cross-sectional study conducted in Qingdao and Weihai, Shandong Province, eastern China. Factors potentially associated with Toxocara infection were identified by logistic regression analysis. The overall Toxocara seroprevalence among the study population (n = 2866) was 12.25%, and a significantly higher seroprevalence in psychiatric patients (16.40%, 73/445) than that in clinically healthy individuals (13.07%, 187/1431) and pregnant women (9.19%, 91/990) was revealed. Univariate analyses suggested that keeping dogs at home (OR = 0.06, 95% CI 0.05–0.08, P<0.001), contact with cats and dogs (OR = 0.42, 95% CI 0.33–0.53, P<0.001) and exposure with soil (OR = 0.37, 95% CI 0.28–0.49, P<0.001) were risk factors associated with Toxocara infection.

Conclusions/Significance

The present study revealed, for the first time, that human infection with Toxocara is common in eastern China, posing a significant public health concern. Increasing human and dog populations, population movements and climate change all will serve to increase the importance of this zoonosis. Further studies under controlled conditions are necessary to define potential morbidity associated with Toxocara infection.     

Risk Factors for Community-Acquired Urinary Tract Infections Caused by ESBL-Producing Enterobacteriaceae –A Case–Control Study in a Low Prevalence Country

Community-acquired urinary tract infection (CA-UTI) is the most common infection caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, but the clinical epidemiology of these infections in low prevalence countries is largely unknown. A population based case-control study was conducted to assess risk factors for CA-UTI caused by ESBL-producing E. coli or K. pneumoniae. The study was carried out in a source population in Eastern Norway, a country with a low prevalence of infections caused by ESBL-producing Enterobacteriaceae. The study population comprised 100 cases and 190 controls with CA-UTI caused by ESBL-producing and non-ESBL-producing E. coli or K. pneumoniae, respectively. The following independent risk factors of ESBL-positive UTIs were identified: Travel to Asia, The Middle East or Africa either during the past six weeks (Odds ratio (OR) = 21; 95% confidence interval (CI): 4.5–97) or during the past 6 weeks to 24 months (OR = 2.3; 95% CI: 1.1–4.4), recent use of fluoroquinolones (OR = 16; 95% CI: 3.2–80) and β-lactams (except mecillinam) (OR = 5.0; 95% CI: 2.1–12), diabetes mellitus (OR = 3.2; 95% CI: 1.0–11) and recreational freshwater swimming the past year (OR = 2.1; 95% CI: 1.0–4.0). Factors associated with decreased risk were increasing number of fish meals per week (OR = 0.68 per fish meal; 95% CI: 0.51–0.90) and age (OR = 0.89 per 5 year increase; 95% CI: 0.82–0.97). In conclusion, we have identified risk factors that elucidate mechanisms and routes for dissemination of ESBL-producing Enterobacteriaceae in a low prevalence country, which can be used to guide appropriate treatment of CA-UTI and targeted infection control measures.     

Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite’s DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13–25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition - with the appearance of ‘tethered’ parasites – malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with T. gondii and inhibited parasite replication early in the first cycle of infection in vitro, highlighting their therapeutic potential for the treatment of toxoplasmosis.     

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63 kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.     

Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.     

Risk of Cerebral Palsy and Childhood Epilepsy Related to Infections before or during Pregnancy

Background and Aim

Maternal infections during pregnancy have been associated with several neurological disorders in the offspring. However, given the lack of specificity for both the exposures and the outcomes, other factors related to infection such as impaired maternal immune function may be involved in the causal pathway. If impaired maternal immune function plays a role, we would expect infection before pregnancy to be associated with these neurological outcomes.

Methods/Principal Findings

The study population included all first-born singletons in Denmark between January 1 1982 and December 31 2004. We identified women who had hospital-recorded infections within the 5 year period before pregnancy, and women who had hospital-recorded infections during pregnancy. We grouped infections into either infections of the genitourinary system, or any other infections. Cox models were used to estimate adjusted hazard ratios (aHRs) with 95% confidence interval (CI). Maternal infection of the genitourinary system during pregnancy was associated with an increased risk of cerebral palsy (aHR = 1.63, 95% CI: 1.34–1.98) and epilepsy (aHR = 1.27, 95% CI: 1.13–1.42) in the children, compared to children of women without infections during pregnancy. Among women without hospital-recorded infections during pregnancy, maternal infection before pregnancy was associated with an increased risk of epilepsy (aHR = 1.35, 95% CI: 1.21–1.50 for infections of the genitourinary system, and HR = 1.12, 95% CI: 1.03–1.22 for any other infections) and a slightly higher risk of cerebral palsy (aHR = 1.20, 95% CI: 0.96–1.49 for infections of the genitourinary system, and HR = 1.23, 95% CI: 1.06–1.43 for any other infections) in the children, compared to children of women without infections before (and during) pregnancy.

Conclusions

These findings indicate that the maternal immune system, maternal infections, or factors related to maternal immune function play a role in the observed associations between maternal infections before pregnancy and cerebral diseases in the offspring.