Performance of Polymerase Chain Reaction Analysis of the Amniotic Fluid of Pregnant Women for Diagnosis of Congenital Toxoplasmosis: A Systematic Review and Meta-Analysis

IntroductionCongenital infection caused by Toxoplasma gondii can cause serious damage that can be diagnosed in utero or at birth, although most infants are asymptomatic at birth. Prenatal diagnosis of congenital toxoplasmosis considerably improves the prognosis and outcome for infected infants. For this reason, an assay for the quick, sensitive, and safe diagnosis of fetal toxoplasmosis is desirable.GoalTo systematically review the performance of polymerase chain reaction (PCR) analysis of the amniotic fluid of pregnant women with recent serological toxoplasmosis diagnoses for the diagnosis of fetal toxoplasmosis.MethodA systematic literature review was conducted via a search of electronic databases; the literature included primary studies of the diagnostic accuracy of PCR analysis of amniotic fluid from pregnant women who seroconverted during pregnancy. The PCR test was compared to a gold standard for diagnosis.ResultsA total of 1.269 summaries were obtained from the electronic database and reviewed, and 20 studies, comprising 4.171 samples, met the established inclusion criteria and were included in the review. The following results were obtained: studies about PCR assays for fetal toxoplasmosis are generally susceptible to bias; reports of the tests’ use lack critical information; the protocols varied among studies; the heterogeneity among studies was concentrated in the tests’ sensitivity; there was evidence that the sensitivity of the tests increases with time, as represented by the trimester; and there was more heterogeneity among studies in which there was more time between maternal diagnosis and fetal testing. The sensitivity of the method, if performed up to five weeks after maternal diagnosis, was 87% and specificity was 99%.ConclusionThe global sensitivity heterogeneity of the PCR test in this review was 66.5% (I²). The tests show low evidence of heterogeneity with a sensitivity of 87% and specificity of 99% when performed up to five weeks after maternal diagnosis. The test has a known performance and could be recommended for use up to five weeks after maternal diagnosis, when there is suspicion of fetal toxoplasmosis.     

Detection of Toxoplasma gondii DNA in milk of dairy cows from southern Brazil

Consumption of unpasteurized cow's milk may be a transmission route for some pathogenic microorganisms, but there is little information about the risk of Toxoplasma gondii infection. Blood and milk samples were collected in a paired and random fashion from 106 dairy cows and bulk-tank milk samples were also collected from each of the six farms, in southern Brazil. Serum anti-T.gondii antibodies (IgG) were detected by an indirect fluorescent antibody test (IFAT) with a cutoff point of 1:64. Nested PCR targeting the ITS1 was performed on milk samples to detect the Sarcocystidae family, confirmed to be T.gondii by Sanger sequencing. The occurrence of anti-T.gondii antibodies in the herds was 14.1%, (15/106) with seropositive cows in all herds. Antibody titers in positive samples ranged from 64 to 128. T.gondii DNA was detected in 2.8% (03/106) of the milk samples. The ITS1 sequences generated in this study were ON809793 - ON809794 and the sequencing revealed 98–100% identity with T. gondii DNA sequences deposited in GenBank. All cows PCR positive for T.gondii in milk were negative for IgG antibodies in serum, suggesting that naturally infected cows may shed T. gondii in milk in the acute phase of infection. The results of this study demonstrate that T. gondii DNA may be detected in raw cow's milk, so the potential risks of lactogenic infection should be considered. The presence of T. gondii DNA in milk does not confirm that the protozoa are viable and infective, and further investigations into the role of cow's milk in the epidemiology of toxoplasmosis are needed.     

Seroprevalence of Toxoplasma gondii infection in slaughter pigs in Shanghai,China

Toxoplasma gondii is a pathogen that seriously threatens the health of humans and animals. However, the current infection status of T. gondii in slaughter pigs in Shanghai is still not clear. To investigate the seroprevalence of T. gondii infection and analyze the prevalence factors associated with the parasite infection, 1158 serum samples were collected from five slaughterhouses in three districts between 2015 and 2018. Serum antibodies against T. gondii were detected in 160 pigs (13.8%) by enzyme-linked immunosorbent assay (ELISA). Additionally, seroprevalence rates differed among different districts (ranging from 4.0% in JD-2 to 17.6% in JD-1), seasons (ranging from 6.7% in winter to 17.8% in autumn), and years (ranging from 8.0% in 2016 to 26.8% in 2015). Region, season, and year were the main factors affecting T. gondii infection in these pigs. There were few reports on serological monitoring of T. gondii in Shanghai slaughterhouses between 2015 and 2018, and the number of infections had steadily increased over the past several consecutive years. Therefore, our data are helpful to understand the epidemic status of T. gondii in Shanghai, which will strengthen the prevention and treatment of swine toxoplasmosis.     

Prevalence of Toxoplasma gondii in Dogs in Zhanjiang,Southern China

Toxoplasmosis, caused by Toxoplasma gondii, is a parasitic zoonosis with worldwide distribution. The present study investigated the prevalence of T. gondii in dogs in Zhanjiang city, southern China, using both serological and molecular detection. A total of 364 serum samples and 432 liver tissue samples were collected from the slaughter house between December 2012 and January 2013 and were examined for T. gondii IgG antibody by ELISA and T. gondii DNA by semi-nested PCR based on B1 gene, respectively. The overall seroprevalence of T. gondii IgG antibody was 51.9%, and T. gondii DNA was detected in 37 of 432 (8.6%) liver tissue samples. These positive DNA samples were analyzed by PCR-RFLP at 3''- and 5''-SAG2. Only 8 samples gave the PCR-RFLP data, and they were all classified as type I, which may suggest that the T. gondii isolates from dogs in Zhanjiang city may represent type I or type I variant. This study revealed the high prevalence of T. gondii infection in dogs in Zhanjiang city, southern China. Integrated measures should be taken to prevent and control toxoplasmosis in dogs in this area for public health concern.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

Toxoplasmosis: stages of the protozoan life cycle and risk assessment in humans and animals for an enhanced awareness and an improved socio-economic status

Toxoplasma gondii is a protozoan parasite distributed globally. It causes toxoplasmosis, which is prevalent in animals, birds, and soil. T. gondii infection leads to severe pathological impacts in immunodeficient patients and congenital cases. This review indicated that high prevalence groups had close contact with cats, dogs, consumed uncooked raw fruits, meat, or vegetables and the socio-economic level noted to be one of the crucial factors that influence toxoplasmosis. Toxoplasmosis infection is high in low-income countries and low in developed European countries. Immunosuppressed groups and pregnant women were the highly vulnerable groups. The epidemiology of the parasite enumerated various routes of infections; but consumption of T. gondii contaminated food was the major route of disease transmission. However, the role of meat and meat-producing animals on disease transmission remained unclear. Unfiltered water acts as the primary reservoir of toxoplasmosis transmission. The diagnostic methods for determining T. gondii infection are not the gold standard, and different approaches have been prescribed to analyze the infected populations based on the organs affected. Although toxoplasmosis was reported before 70 years, no appropriate solution noted to be recommended to treat this disease. Based on the present analyses, it concluded that the eradication of toxoplasmosis would be challenging from the world until people''s socio-economic level is improved. The main aim of the present study was to analyze and update the disease transmission, epidemiology, and possible clinical interventions of toxoplasmosis.     

Gefitinib Inhibits the Growth of Toxoplasma gondii in HeLa Cells

Toxoplasma gondii is the causative agent of toxoplasmosis with symptoms of congenital neurological and ocular diseases and acquired lymphadenitis, retinochoroiditis, and meningoencephalitis. Small molecules which block the activity of protein kinases were tested in in vitro culture of T. gondii to find new therapeutic drugs of safer and more effective than the combined administration of pyrimethamine and sulfadoxine that sometimes provoke lethal Stevens-Johnson syndrome. Among them, Gefitinib and Crizotinib inhibited intracellular growth of T. gondii in HeLa cells by counting the number of T. gondii per parasitophorous vacuolar membrane whereas Sunitinib did not. Gefitinib inhibited the growth of T. gondii in a dose-dependent manner over 5 µM up to the tolerable concentration of HeLa cells and halted the division of the parasite immediately from the time point of treatment. Gefitinib inhibition suggests that tyrosine kinases of EGFR family or other homologous kinases of the parasite itself may be the target to cause the block of T. gondii growth.     

Toxoplasma gondii: The effects of infection at different stages of pregnancy on the offspring of mice

Congenital toxoplasmosis can cause fetal damage in humans and domestic animals. This study was focused on the effects of Toxoplasma gondii (Prugniaud strain) infection at different stages of pregnancy on the offspring of mice. Results showed that newborn mice from all infected groups were significantly lower in weight than those from the control group but significant difference was not found among these groups at day 60 after birth. The survival rate of the offspring from the group of mice infected at the earlier stage of pregnancy was significantly lower than those of infected and control groups. The positive offspring (with cysts found in their brain tissues) born from the mice infected at the earlier and intermediate stages of pregnancy showed a shorter latency and greater number of errors in the step-through passive avoidance test than those born from the mice infected at the late stage of pregnancy, the control group and the negative offspring from the infected groups. The number of cysts in the brain tissue was significantly higher in the offspring born from the groups of mice infected at the earlier and intermediate stages of pregnancy than those from the group of mice infected at the late stage of pregnancy. In addition, our results indicated that a high congenital transmission rate (90%) occurred in this NIH mouse model. In conclusion, the earlier and intermediate maternal infection of T. gondii can result in severe congenital toxoplasmosis, exhibiting conditions such as stillbirth or non-viability, and learning or memory capability damage in this mouse model. These results not only provide useful data for better understanding the effects of T. gondii infection on the offspring of mice infected at different stages of pregnancy but also for better consideration of the effect of this infection on other mammalian hosts including humans.     

Effects of Toxoplasma gondii (Gleadle strain) on the host-parasite relationship in trichinosis.

The effects of concurrent infection with Toxoplasma gondii on the host-parasite relationship in trichinosis were studied. Infected mice showed a delay in expulsion of Trichinella spiralis adults from the gut. Persisting adult female worms were fecund but the numbers of larvae recovered from the muscles were not increased. Increased resistance to the systemic phase of trichinosis was shown by reduced numbers of muscle larvae after intravenous injection of newborn larvae in animals with toxoplasmosis as compared with control mice. There were no differences in small bowel pathology of trichinous mice with and without toxoplasmosis but inflammation around muscle cysts of T. spiralis was reduced in mice with toxoplasmosis. The eosinophilia which normally develops in mice with trichinosis was suppressed by concurrent toxoplasmosis. Trichinella infection did not alter the numbers of T. gondii cysts recovered from the brain 4 weeks after infection. It is suggested that the delay in expulsion of adult worms, decrease in muscle inflammation around T. spiralis cysts, and inhibition of eosinophilia result from immune suppression, while the reduction in numbers of muscle larvae after intravenous injection of newborn larvae reflects enhanced nonspecific resistance to infection in toxoplasmosis.     

A Highly Conserved Toxo1 Haplotype Directs Resistance to Toxoplasmosis and Its Associated Caspase-1 Dependent Killing of Parasite and Host Macrophage

Natural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1β. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-β secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance.     

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.     

The Dysfunction of CD4+CD25+ Regulatory T Cells Contributes to the Abortion of Mice Caused by Toxoplasma gondii Excreted-Secreted Antigens in Early Pregnancy

Toxoplasma gondii is an opportunistic intracellular parasite that is highly prevalent in human and warm-blooded animals throughout the world, leading to potentially severe congenital infections. Although the abortion caused by T. gondii is believed to be dependent on the timing of maternal infection during pregnancy, the mechanism remains unclear. This study was focused on the effects of T. gondii excreted-secreted antigens on pregnant outcomes and CD4⁺CD25⁺ Foxp3⁺ regulatory T cells at different stages of pregnancy. The results showed that in mice the frequency and suppressive function of CD4⁺CD25⁺ regulatory cells were diminished after injection of T. gondii excreted-secreted antigens at early and intermediate stages of pregnancy. The abortion caused by T. gondii excreted-secreted antigens at early pregnancy could be partly prevented by adoptively transferring of CD4⁺CD25⁺ cells from the mice injected with T. gondii excreted-secreted antigens at late pregnancy, but not from the mice with the same treatment at early pregnancy. Furthermore, T. gondii excreted-secreted antigens induced apoptosis of CD4⁺CD25⁺ regulatory cells of mice in early and intermediate stages of pregnancy by down-regulating their Bcl-2 expressions and Bcl-2/Bax ratio. This study provides new insights into the mechanism that T. gondii infection is the high risk factor for abortion in early pregnancy.     

Toxoplasma gondii infection/exposure and the risk of brain tumors: A systematic review and meta-analysis

BackgroundBrain tumors are among the most fatal cancers with substantial morbidity and mortality worldwide. Epidemiologic evidence suggests that infectious agents, especially, protozoan parasite Toxoplasma gondii could be a possible risk factor or contributor. Here, we performed a systematic review and meta-analysis to evaluate the possible association between T. gondii infection/exposure and risk of brain tumors.MethodsWe searched the PubMed, Embase, Scopus, and Web of Science collection databases from inception through 1st of December 2021. Pooled estimates of odds ratios (ORs) and 95% confidence intervals (CIs) were generated using random effects models. We did the subgroup analysis according to tumor types. Statistical tests for heterogeneity and sensitivity analyses were applied.ResultsA total of seven eligible studies comprising 2323 patients diagnosed with brain tumors and 5131 healthy controls were included in the meta-analysis. T. gondii infection/exposure prevalence was 24.2% (95%CI, 12.7%–41.2) in cases and 12.9% (95%CI, 7.0–22.6%) in control subjects. Pooled analysis showed an overall OR of 1.96 (95%CI, 1.37–2.80), indicating a significant increased risk of brain tumors associated with T. gondii infection/exposure. In subgroup analysis T. gondii infection/exposure was significantly associated with gliomas (OR: 1.64, 95%CI, 1.15–2.33), meningioma (OR: 2.30, 95%CI, 1.0–5.27) and other types of brain tumors (OR: 2.19, 95%CI, 1.02–4.71).ConclusionThis study provides suggestive evidence for an association between T. gondii infection/exposure and brain tumors. Our findings should be further confirmed by well-designed cohort studies with strict control of confounders. Moreover, we suggest that future studies also focus on the effect of T. gondii infection/exposure to the types of brain tumors.     

Increased CD11b+ Gr‐1+ cell population in the placenta after infection with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan pathogen that can cross the placenta, resulting in congenital toxoplasmosis with severe fetal brain abnormalities. The molecular mechanisms of immune responses against T. gondii infection in the placenta have largely remained unclear. An analytical method for characterizing phenotypes of immune cells in the placenta by flow cytometry was established and it was found that numbers of CD11b⁺ Gr‐1⁺ cells in the placenta increased significantly after T. gondii infection. These results suggest that innate immune responses play an important role in immunity against T. gondii infection via the feto‐maternal interface.     

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.     

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5''and 3''ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.     

Isolation of Toxoplasma gondii strains similar to Africa 1 genotype in Turkey

IntroductionToxoplasma gondii is a protozoon parasite that has a worldwide dissemination. It can cause serious clinical problems such as congenital toxoplasmosis, retinochoroiditis, and encephalitis. Currently, T. gondii genotypes are being associated with these clinical presentations which may help clinicians design their treatment strategy.Case reportsTwo T. gondii strains named Ankara and Ege-1 were isolated from newborns with congenital toxoplasmosis in Central and Western Anatolia, respectively. Ankara and Ege-1 strains were isolated from the cerebrospinal fluid of newborns. According to microsatellite analysis, Ankara and Ege-1 strains were sorted as Africa 1 genotype.ConclusionT. gondii strains isolated in Turkey were first time genotyped in this study. Africa 1 genotype has previously been isolated in immunosuppressed patients originating from sub-Saharan Africa. The reason of detecting a strain mainly detected in Africa can be associated with Turkey's specific geographical location. Turkey is like a bridge between Asia, Europe and Africa. Historically, Anatolia was on the Silk Road and other trading routes that ended in Europe. Thus, detecting Africa 1 strain in Anatolia can be anticipated. Consequently, strains detected mainly in Europe and Asia may also be detected in Anatolia and vice versa. Therefore, further studies are required to isolate more strains from Turkey.     

TGF-β in Toxoplasmosis: Friend or foe?

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan causing several forms of toxoplasmosis in humans. The main mechanisms that allow the development of the prolonged forms of the disease and its subsequent pathology are yet to be clarified. However, many researchers have hypothesized that immunological and genetic parameters may play crucial roles in the etiology of the disease. Transforming growth factor beta (TGF-β) is a cytokine with a dual role in the regulation of immune responses including those against parasites. However, the relationship between TGF-β and immune responses against T .gondii are not fully understood. The important roles played by TGF-β in the development of Th17 and T regulatory lymphocytes, mucosal immunity and regulation of immune responses have been documented and this provides insights into TGF-β function during parasitic infections such as toxoplasmosis. Therefore, the aim of this review is to collate the current information regarding the status and association of TGF-β with T. gondii infection.     

Analysis of Toxoplasma gondii surface antigen 2 gene (SAG2). Relevance of genotype I in clinical toxoplasmosis

Toxoplasma gondii is one of the most successful protozoan parasites given its ability to manipulate the immune system and establish a chronic infection. It is a parasite with a significant impact on human health, mainly in immunocompromised patients. In Europe and North America, only a few clonal genotypes (I, II and III) seem to be responsible for the vast majority of Toxoplasma infections. Surface antigen 2 gene (SAG2) has been extensively used for genotyping T. gondii isolates. The analysis of this locus reveals that in Northern hemisphere, human disease causing isolates are mainly type II, whereas T. gondii isolated from different animals are both type II and III. Since the immune response depends on parasite genotype, it seems relevant to characterize parasites producing human toxoplasmosis in different geographical areas. The growing information about the prevalent T. gondii genotypes in South America mostly refers to domestic animals. This is the first report of genetic characterization of T. gondii isolates from clinical samples in Chile, South America. All the samples analyzed corresponded to SAG2 type I isolates, and they differ from classic SAG2 type I by genetic polymorphisms. This study contributes to the scarce available information on T. gondii at South America, and reinforces an emerging concept suggesting that SAG2 type I, rather than II, parasites are a frequent cause of clinical toxoplasmosis in this continent.