Characterization of ROP18 alleles in human toxoplasmosis

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis.     

Toxoplasma gondii: P30 peptides recognition pattern in human toxoplasmosis

In this study, human sera reactivity against nine peptides derived from the Toxoplasma gondii P30 protein was assessed by ELISA in patients with different clinical forms of toxoplasmosis. Same as has been reported in mice, sera from congenital, ocular and chronic asymptomatic toxoplasmosis patients recognized more strongly peptides from the protein’s carboxy-terminus, being peptide 2017 (amino acids 301-320) the one most strongly recognized by sera from patients with ocular toxoplasmosis. Serum samples collected from 13 patients without ocular infection, 13 with inactive chorioretinal scars, 6 with active ocular infection and 10 seronegative individuals were then screened for anti-2017 IgG. Peptide 2017 was recognized by all patients’ samples but not by sera from T. gondii-seronegative individuals. No statistically significant differences were found between the absorbance levels of groups with and without lesions or with active or inactive ocular lesions, as determined by ANOVA.     

Contrasting Effect of Prepulse Signals on Performance of Toxoplasma-Infected and Toxoplasma-Free Subjects in an Acoustic Reaction Times Test

Background

About 30% of people on Earth have latent toxoplasmosis. Infected subjects do not express any clinical symptoms, however, they carry dormant stages of parasite Toxoplasma for the rest of their life. This form of toxoplasmosis is mostly considered harmless, however, recent studies showed its specific effects on physiology, behaviour and its associations with various diseases, including psychiatric disorders such as schizophrenia. Individuals who suffer from schizophrenia have about 2.7 times higher prevalence of Toxoplasma-seropositivity than controls, which suggests that some traits characteristic of schizophrenic patients, including the sex difference in schizophrenia onset, decrease of grey matter density in specific brain areas and modification of prepulse inhibition of startle reaction could in fact be caused by toxoplasmosis for those patients who are Toxoplasma-seropositive.

Methodology/Principal Findings

We measured the effect of prepulse inhibition/facilitation of the startle reaction on reaction times. The students, 170 women and 66 men, were asked to react as quickly as possible to a startling acoustic signal by pressing a computer mouse button. Some of the startling signals were without the prepulse, some were 20 msec. preceded by a short (20 msec.) prepulse signal of lower intensity. Toxoplasma-seropositive subjects had longer reaction times than the controls. Acoustic prepulse shorted the reaction times in all subjects. This effect of prepulse on reaction times was stronger in male subjects and increased with the duration of infection, suggesting that it represented a cumulative effect of latent toxoplasmosis, rather than a fading out after effect of past acute toxoplasmosis.

Conclusions

Different sensitivity of Toxoplasma-seropositive and Toxoplasma-seronegative subjects on effect of prepulses on reaction times (the toxoplasmosis-prepulse interaction) suggested, but of course did not prove, that the alternations of prepulse inhibition of startle reaction observed in schizophrenia patients probably joined the list of schizophrenia symptoms that are in fact caused by latent toxoplasmosis.     

First Colombian multicentric newborn screening for congenital toxoplasmosis

Aims

To determine the incidence of congenital toxoplasmosis in Colombian newborns from 19 hospital or maternal child health services from seven different cities of five natural geographic regions (Caribbean, Central, Andean, Amazonia and Eastern).

Materials and Methods

We collected 15,333 samples from umbilical cord blood between the period of March 2009 to May 2010 in 19 different hospitals and maternal-child health services from seven different cities. We applied an IgM ELISA assay (Vircell, Spain) to determine the frequency of IgM anti Toxoplasma. The results in blood cord samples were confirmed either by western blot and repeated ELISA IgM assay. In a sub-sample of 1,613 children that were negative by the anti-Toxoplasma IgM assay, the frequency of specific anti-Toxoplasma IgA by the ISAGA assay was determined. All children with positive samples by IgM, IgA, clinical diagnosis or treatment during pregnancy were recalled for confirmatory tests after day 10 of life.

Results

61 positive samples for specific IgM (0.39%) and 9 positives for IgA (0.5%) were found. 143 questionnaires were positive for a clinical diagnosis or treatment for toxoplasmosis during pregnancy. 109 out of the 218 children that had some of the criteria for postnatal confirmatory tests were followed. Congenital toxoplasmosis infection was confirmed in 15 children: 7 were symptomatic, and three of them died before the first month of life (20% of lethality). A significant correlation was found between a high incidence of markers for congenital toxoplasmosis and higher mean annual rainfall for the city.

Conclusions

Incidence for congenital toxoplasmosis is significantly different between hospitals or maternal child health services from different cities in Colombia. Mean annual rainfall was correlated with incidence of congenital toxoplasmosis.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

Interferon-Gamma Release Assay: An Effective Tool to Detect Early Toxoplasma gondii Infection in Mice

Early diagnosis of Toxoplasma gondii infection before the formation of tissue cysts is vital for treatment, as drugs available for toxoplasmosis cannot kill bradyzoites contained in the cysts. However, current methods, such as antibody-based ELISA, are ineffective for detection of early infection. Here, we developed an interferon-gamma release assay (IGRA), measuring the IFN-γ released by T lymphocytes stimulated by Toxoplasma antigen peptides in vitro, for the detection of T. gondii infection in mice. Splenocytes isolated from infected mice were stimulated by peptides derived from dense granule proteins GRA4 and GRA6 and rhoptry protein ROP7, and released IFN-γ was measured by ELISA. Results showed that both acute and chronic infection could be detected by IGRA. More importantly, IGRA detected infection as early as the third day post infection; while serum IgM and IgG were detected 9 days and 13 days post infection, respectively. Our findings demonstrated that an IGRA-positive and ELISA-negative sample revealed an early infection, indicating the combination of IGRA and ELISA can be employed for the early diagnosis of T. gondii infection in human beings, cats and livestock.     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Immune Response to Toxoplasma gondii

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [³H]methylated thymidine. Analysis of Toxoplasma-specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma-specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma-specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.     

Toxoplasma gondii: Congenital transmission in a hamster model

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non-invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n = 168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated. Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.     

Effects of Latent Toxoplasmosis on Autoimmune Thyroid Diseases in Pregnancy

Background

Toxoplasmosis, one of the most common zoonotic diseases worldwide, can induce various hormonal and behavioural alterations in infected hosts, and its most common form, latent toxoplasmosis, influences the course of pregnancy. Autoimmune thyroid diseases (AITD) belong to the well-defined risk factors for adverse pregnancy outcomes. The aim of this study was to investigate whether there is a link between latent toxoplasmosis and maternal AITD in pregnancy.

Methods

Cross-sectional study in 1248 consecutive pregnant women in the 9–12^th gestational weeks. Serum thyroid-stimulating hormone (TSH), thyroperoxidase antibodies (TPOAb), and free thyroxine (FT4) were assessed by chemiluminescence; the Toxoplasma status was detected by the complement fixation test (CFT) and anti-Toxoplasma IgG enzyme-linked immunosorbent assay (ELISA).

Results

Overall, 22.5% of the women were positive for latent toxoplasmosis and 14.7% were screened positive for AITD. Women with latent toxoplasmosis had more often highly elevated TPOAb than the Toxoplasma-negative ones (p = 0.004), and latent toxoplasmosis was associated with decrease in serum TSH levels (p = 0.049). Moreover, we found a positive correlation between FT4 and the index of positivity for anti-Toxoplasma IgG antibodies (p = 0.033), which was even stronger in the TPOAb-positive Toxoplasma-positive women, (p = 0.014), as well as a positive correlation between FT4 and log₂ CFT (p = 0.009).

Conclusions

Latent toxoplasmosis was associated with a mild increase in thyroid hormone production in pregnancy. The observed Toxoplasma-associated changes in the parameters of AITD are mild and do not seem to be clinically relevant; however, they could provide new clues to the complex pathogenesis of autoimmune thyroid diseases.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Severe South American Ocular Toxoplasmosis Is Associated with Decreased Ifn-γ/Il-17a and Increased Il-6/Il-13 Intraocular Levels

In a cross sectional study, 19 French and 23 Colombian cases of confirmed active ocular toxoplasmosis (OT) were evaluated. The objective was to compare clinical, parasitological and immunological responses and relate them to the infecting strains. A complete ocular examination was performed in each patient. The infecting strain was characterized by genotyping when intraocular Toxoplasma DNA was detectable, as well as by peptide-specific serotyping for each patient. To characterize the immune response, we assessed Toxoplasma protein recognition patterns by intraocular antibodies and the intraocular profile of cytokines, chemokines and growth factors. Significant differences were found for size of active lesions, unilateral macular involvement, unilateral visual impairment, vitreous inflammation, synechiae, and vasculitis, with higher values observed throughout for Colombian patients. Multilocus PCR-DNA sequence genotyping was only successful in three Colombian patients revealing one type I and two atypical strains. The Colombian OT patients possessed heterogeneous atypical serotypes whereas the French were uniformly reactive to type II strain peptides. The protein patterns recognized by intraocular antibodies and the cytokine patterns were strikingly different between the two populations. Intraocular IFN-γ and IL-17 expression was lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients. Our results are consistent with the hypothesis that South American strains may cause more severe OT due to an inhibition of the protective effect of IFN-γ.     

Direct evidence of Toxoplasma-induced changes in serum testosterone in mice

Latent toxoplasmosis is known to influence the morphology of infected persons and also increases the probability of the birth of male offspring in both humans and mice. All these traits can be related to the observed differences in the concentration of testosterone between Toxoplasma-infected and Toxoplasma-free subjects. However, it is not possible to decide, using the Toxoplasma-human model, whether toxoplasmosis influences the level of testosterone in the infected host or whether individuals with different levels of testosterone vary in the probability of toxoplasma infection. Here we studied changes in the testosterone levels in the latent phase of toxoplasmosis in laboratory mice artificially infected with cystogenic but relatively virulent strain T38 of T. gondii. We observed decreased testosterone levels in both female and male mice with latent toxoplasmosis in comparison to uninfected controls (P = 0.001). The present results indicate that Toxoplasma infection changes the concentration of serum testosterone in mice and human rather than changed concentration of testosterone influences the probability of the Toxoplasma infection. It is possible that the decrease of testosterone is an adaptive mechanism of infected mice aimed to compensate toxoplasmosis-induced immunosuppression observed during latent Toxoplasma infection.     

Diagnostic Value of a Rec-ELISA Using Toxoplasma gondii Recombinant SporoSAG,BAG1, and GRA1 Proteins in Murine Models Infected Orally with Tissue Cysts and Oocysts

Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P<0.01). The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05). The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01). The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05). The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05) and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10–40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.     

Survey of the Parasite Toxoplasma gondii in Human Consumed Ovine Meat in Tunis City

Toxoplasmosis has been recognized as parasitic zoonosis with the highest human incidence. The human infection by the parasite can lead to severe clinical manifestations in congenital toxoplasmosis and immunocompromised patients. Contamination occurs mainly by foodborne ways especially consumption of raw or undercooked meat. In contrast to other foodborne infections, toxoplasmosis is a chronic infection which would make its economic and social impact much higher than even previously anticipated. Ovine meat was advanced as a major risk factor, so we investigated its parasite survey, under natural conditions. Serological MAT technique and touchdown PCR approaches were used for prevalence determination of the parasite in slaughtered sheep intended to human consumption in Tunis City. The genotyping was carried by SNPs analysis of SAG3 marker. Anti-Toxoplasma antibodies were present in 38.2% of young sheep and in 73.6% of adult sheep. Molecular detection revealed the contamination of 50% of ewes’ tissue. Sequencing and SNPs analysis enabled unambiguous typing of meat isolates and revealed the presence of mixed strains as those previously identified from clinical samples in the same area. Our findings conclude that slaughtered sheep are highly infected, suggesting them as a major risk factor of Toxoplasma gondii transmission by meat consumption. Special aware should target consequently this factor when recommendations have to be established by the health care commanders.     

Fatal attraction phenomenon in humans: cat odour attractiveness increased for toxoplasma-infected men while decreased for infected women

Background

Latent toxoplasmosis, a lifelong infection with the protozoan Toxoplasma gondii, has cumulative effects on the behaviour of hosts, including humans. The most impressive effect of toxoplasmosis is the “fatal attraction phenomenon,” the conversion of innate fear of cat odour into attraction to cat odour in infected rodents. While most behavioural effects of toxoplasmosis were confirmed also in humans, neither the fatal attraction phenomenon nor any toxoplasmosis-associated changes in olfactory functions have been searched for in them.

Principal Findings

Thirty-four Toxoplasma-infected and 134 noninfected students rated the odour of urine samples from cat, horse, tiger, brown hyena and dog for intensity and pleasantness. The raters were blind to their infection status and identity of the samples. No signs of changed sensitivity of olfaction were observed. However, we found a strong, gender dependent effect of toxoplasmosis on the pleasantness attributed to cat urine odour (p = 0.0025). Infected men rated this odour as more pleasant than did the noninfected men, while infected women rated the same odour as less pleasant than did noninfected women. Toxoplasmosis did not affect how subjects rated the pleasantness of any other animal species'' urine odour; however, a non-significant trend in the same directions was observed for hyena urine.

Conclusions

The absence of the effects of toxoplasmosis on the odour pleasantness score attributed to large cats would suggest that the amino acid felinine could be responsible for the fatal attraction phenomenon. Our results also raise the possibility that the odour-specific threshold deficits observed in schizophrenia patients could be caused by increased prevalence of Toxoplasma-infected subjects in this population rather than by schizophrenia itself. The trend observed with the hyena urine sample suggests that this carnivore, and other representatives of the Feliformia suborder, should be studied for their possible role as definitive hosts in the life cycle of Toxoplasma.     

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2?μM to as low as 110?nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5?brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.     

Toxoplasmosis-Associated Difference in Intelligence and Personality in Men Depends on Their Rhesus Blood Group but Not ABO Blood Group

Background

The parasite Toxoplasma gondii influences the behaviour of infected animals and probably also personality of infected humans. Subjects with a Rhesus-positive blood group are protected against certain behavioural effects associated with Toxoplasma infection, including the deterioration of reaction times and personality factor shift.

Methodology/Principal Findings

Here, we searched for differences in the toxoplasmosis-associated effects between RhD-positive and RhD-negative subjects by testing 502 soldiers with two personality tests and two intelligence tests. The infected subjects expressed lower levels of all potentially pathognomic factors measured with the N-70 questionnaire and in neurasthenia measured with NEO-PI-R. The RhD-positive, Toxoplasma-infected subjects expressed lower while RhD-negative, Toxoplasma-infected subjects expressed higher intelligence than their Toxoplasma-free peers. The observed Toxoplasma-associated differences were always larger in RhD-negative than in RhD-positive subjects.

Conclusions

RhD phenotype plays an important role in the strength and direction of association between latent toxoplasmosis and not only psychomotor performance, but also personality and intelligence.     

Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.     

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.