ABSTRACTToxoplasma gondii is a highly prevalent pathogen causing zoonotic infections with significant public health implications. Yet, our understanding of long-term consequences, associated risk factors, and the potential role of co-infections is still limited. Seroepidemiological studies are a valuable approach to address open questions and enhance our insights into T. gondii across human populations. Here, we present substantial advancements to our previously developed T. gondii multiplex serology assay, which is based on the immunodominant antigens SAG1 and P22. While our previous bead-based assay quantified antibody levels against multiple targets in a high-throughput fashion requiring only a small sample volume, impaired assay characteristics emerged in sample dilutions beyond 1:100 and when being transferred to magnetic beads. Both are now critical for inclusion in large-scale seroprevalence studies. Using the truncated versions, SAG1D1 and P22trunc, significantly enhanced signal-to-noise ratios were achieved with almost perfect concordance with the gold-standard Sabin–Feldman dye test. In sample dilutions of 1:100, the diagnostic accuracy of SAG1D1 and P22trunc reached sensitivities (true positive rates) of 98% and 94% and specificities (true negative rates) of 93% and 95%, respectively. Importantly, performance metrics were reproducible in a 1:1,000 sample dilution, using both magnetic and nonmagnetic beads. Thresholds for seropositivity were derived from finite mixture models and performed equally well as thresholds by receiver operating characteristic analysis. Our improved multiplex serology assay is therefore able to generate robust and reproducible performance metrics under various assay conditions. Inclusion of T. gondii antibody measurements with other pathogens, in multiplex serology panels will allow for large-scale seroepidemiological research.IMPORTANCEToxoplasma gondii is a pathogen of significant public health concern due to its widespread prevalence and zoonotic potential. However, our understanding of key aspects, such as risk factors for infection and disease, potential outcomes, and their trends, remains limited. Seroepidemiological studies in large cohorts are invaluable for addressing these questions but remain scarce. Our revised multiplex serology assay equips researchers with a powerful tool capable of delivering T. gondii serum antibody measurements with high sensitivity and specificity under diverse assay conditions. This advancement paves the way for the integration of T. gondii antibody measurements into multi-pathogen multiplex serology panels, promising valuable insights into public health and pathogen interactions.

Background: Toxoplasmosis and cytomegalovirus (CMV) congenital infection present with similar clinical pictures. Both infections have long-term sequelae that can be mitigated by early detection and treatment. Coinfection is uncommonly reported. Methods: Dichorionic diamniotic twins born at 35 weeks of gestation were investigated for congenital infections due to abnormalities on the antenatal scan at 31 weeks of gestation. Antenatal investigations were delayed due to late booking and delay in maternal investigations. In the neonatal period, they suffered discordant symptoms and were both investigated for Toxoplasma gondii infection. This diagnosis was confirmed in twin 2 but proved difficult in twin 1 who had a weakly positive polymerase chain reaction with inconclusive serology. Twin 1 was also diagnosed with congenital CMV, further complicating the clinical picture. Toxoplasmosis can cause long-term sequelae, and definitive diagnosis requires serology at 12 months of age; in view of this, treatment for congenital toxoplasmosis was initiated in both twins. Twin 1 was also treated for congenital CMV. Results: Due to limitations in serological investigations in neonates, diagnosing congenital toxoplasmosis can be challenging, and initiating treatment may be warranted in suspected cases, given the risk of infective complications. Discordant presentations between twins are known in congenital toxoplasmosis and CMV, but coinfection has rarely been reported without concurrent immunocompromise. A high index of suspicion should be maintained in the twin of an infected neonate, and the possibility of multiple infections should be considered. Multidisciplinary working is crucial in reaching a diagnosis and treating appropriately.

BackgroundThe parasite Toxoplasma gondii can cause congenital toxoplasmosis following primary infection in a pregnant woman. It is therefore important to distinguish between recent and past infection when both T. gondii-specific IgM and IgG are detected in a single serum in pregnant women. Toxoplasma gondii-specific IgG avidity testing is an essential tool to help to date the infection. However, interpretation of its results can be complex.ObjectivesTo review the benefits and limitations of T. gondii-specific avidity testing in pregnant women, to help practitioners to interpret the results and adapt the patient management.SourcesPubMed search with the keywords avidity, toxoplasmosis and Toxoplasma gondii for articles published from 1989 to 2019.ContentToxoplasma gondii-specific IgG avidity testing remains a key tool for dating a T. gondii infection in immunocompetent pregnant women. Several commercial assays are available and display comparable performances. A high avidity result obtained on a first-trimester serum sample is indicative of a past infection, which occurred before pregnancy. To date, a low avidity result must still be considered as non-informative to date the infection, although some authors suggest that very low avidity results are highly suggestive of recent infections depending on the assay. Interpretation of low or grey zone avidity results on a first-trimester serum sample, as well as any avidity result on a second-trimester or third-trimester serum sample, is more complex and requires recourse to expert toxoplasmosis laboratories.ImplicationsAlthough used for about 30 years, T. gondii-specific avidity testing has scarcely evolved. The same difficulties in interpretation have persisted over the years. Some authors have proposed additional thresholds to exclude an infection of <9 months, or in contrast to confirm a recent infection. Such thresholds would be of great interest to adapt management of pregnant women and avoid unnecessary treatment; however, they need confirmation and further studies.

Congenital toxoplasmosis occurs following transplacental transfer of Toxoplasma gondii Irrespective of symptom status at birth, infants with congenital infection may develop serious long-term sequelae, including learning disability, seizures, hydrocephalus, motor and hearing deficits, chorioretinitis and retinal scarring with impaired vision. Timely diagnosis facilitates early initiation of therapy, aimed at prevention or amelioration of adverse clinical consequences. Diagnosis can be difficult, however, since acutely infected mothers are often asymptomatic and laboratory testing can be complex. Moreover, any decision to start treatment in the newborn must include careful consideration of the benefits and risks. This paper outlines a structured approach for managing an infant born to a woman with possible or confirmed T. gondii infection during pregnancy, including key aspects of the antenatal history, interpretation and timing of investigations, treatment and appropriate follow-up. Our recommendations are based on current evidence in the literature, consensus from two UK paediatric infectious disease centres and the UK specialist Toxoplasma Reference Unit.

•Immunosuppressive medications are often under-recognised risk factors for toxoplasmosis in non-HIV persons.•This is the first described case of cerebral toxoplasmosis developing on MMF monotherapy, with six other published cases developing on MMF with other concurrent immunosuppressant medication.•This case highlights the importance of considering cerebral toxoplasmosis in non-HIV persons on immunosuppressive therapy for inflammatory neuromuscular disorders.

Multiplex Serology is a high-throughput technology developed to simultaneously measure specific serum antibodies against multiple pathogens in one reaction vessel. Serological assays for hepatitis B (HBV) and C (HCV) viruses, human T-lymphotropic virus 1 (HTLV-1) and the protozoan parasite Toxoplasma gondii (T. gondii) were developed and validated against established reference assays. For each pathogen, between 3 and 5 specific antigens were recombinantly expressed as GST-tag fusion proteins in Escherichia coli and tested in Monoplex Serology, i.e. assays restricted to the antigens from one particular pathogen. For each of the four pathogen-specific Monoplex assays, overall seropositivity was defined using two pathogen-specific antigens. In the case of HBV Monoplex Serology, the detection of past natural HBV infection was validated based on two independent reference panels resulting in sensitivities of 92.3% and 93.0%, and specificities of 100% in both panels. Validation of HCV and HTLV-1 Monoplex Serology resulted in sensitivities of 98.0% and 95.0%, and specificities of 96.2% and 100.0%, respectively. The Monoplex Serology assay for T. gondii was validated with a sensitivity of 91.2% and specificity of 92.0%. The developed Monoplex Serology assays largely retained their characteristics when they were included in a multiplex panel (i.e. Multiplex Serology), containing additional antigens from a broad range of other pathogens. Thus HBV, HCV, HTLV-1 and T. gondii Monoplex Serology assays can efficiently be incorporated into Multiplex Serology panels tailored for application in seroepidemiological studies.

Transplantation activity is increasing, leading to a growing number of patients at risk for toxoplasmosis. We reviewed toxoplasmosis prevention practices, prevalence, and outcomes for hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT; heart, kidney, or liver) patients in Europe. We collected electronic data on the transplant population and prevention guidelines/regulations and clinical data on toxoplasmosis cases diagnosed during 2010-2014. Serologic pretransplant screening of allo-hematopoietic stem cell donors was performed in 80% of countries, screening of organ donors in 100%. SOT recipients were systematically screened in 6 countries. Targeted anti-Toxoplasma chemoprophylaxis was heterogeneous. A total of 87 toxoplasmosis cases were recorded (58 allo-HSCTs, 29 SOTs). The 6-month survival rate was lower among Toxoplasma-seropositive recipients and among allo-hematopoietic stem cell and liver recipients. Chemoprophylaxis improved outcomes for SOT recipients. Toxoplasmosis remains associated with high mortality rates among transplant recipients. Guidelines are urgently needed to standardize prophylactic regimens and optimize patient management.

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Sixteen laboratories carried out a collaborative study to validate 13/132 as a replacement International Standard (IS) for TOXM (3rd IS for anti-Toxoplasma Serum, Human, 1000 IU). 13/132 is a freeze dried preparation of pooled human plasma from six donors who experienced a recent Toxoplasma gondii infection. The potency of 13/132 was compared to TOXM and 01/600 (1st IS for anti-Toxoplasma IgG, Human, 20 IU). Samples were tested for IgA, IgG, IgG avidity and IgM in agglutination assays; enzyme linked immunosorbent assays (ELISA), enzyme linked fluorescent assays, immunoblots, immunofluorescence assays and the Sabin-Feldman dye test for Ig. 13/132 was strongly positive for Ig, IgA, IgG and IgM and the reproducibility was very good. 13/132 contains high levels of anti-Toxoplasma Ig, IgG and IgM and its potency falls between TOXM and 01/600. The avidity of IgG was found to be low, similar to the avidity of IgG from TOXM. 13/132 was established by the Expert Committee on Biological Standardization as the 4th IS for Antibodies, Human, to T gondii with an assigned unitage of 160 IU per ampoule for Ig by dye test and 263 U per ampoule for IgG by ELISA. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of International Alliance for Biological Standardization.

BACKGROUND:Toxoplasma gondii is found on all continents and can infect all endothermic vertebrates. Toxoplasmosis is a globally important zoonosis with potentially devastating health impacts both for humans and a range of domestic and wild species. The World Health Organisation have repeatedly recommended the collection of accurate epidemiological data for T. gondii, yet despite recognised links between infection of wildlife, domestic animals and humans, seroprevalence in wild species is rarely monitored. Here, serological investigation using the Gold Standard Sabin-Feldman Dye Test was used to test for T. gondii in Eurasian otters (Lutra lutra) found dead, mainly as road-kill, in England and Wales. This is the first spatially widespread study of T. gondii in UK wildlife, and the first extensive survey of T. gondii in Eurasian otters, a sentinel species of fresh waters.FINDINGS:Infection was both common (39.5% prevalence, n = 271) and widespread, with significantly more infection in the east than the west of the UK. There was an increase in seroprevalence with age, but no sex bias.CONCLUSIONS:The relatively high prevalence of T. gondii in a predominantly piscivorous freshwater mammal suggests widespread faecal contamination of freshwater ecosystems with oocysts. Continued surveillance of the Eurasian otter for T. gondii is valuable because of conservation concerns due to the otter's 'near threatened' status on the IUCN Red List and because of the host's role as a sentinel for freshwater health.

The diagnosis of Toxoplasma infection or disease in hematopoietic stem cell transplantation (HSCT) patients is achieved mainly by PCR screening; however screening did not find wide field of use in practice due to costly expenditures of PCR. This study aimed to determine patients at high risk of Toxoplasma infection or disease before transplantation by stem cell originated buffy coat PCR and subsequently to screen them. Buffy coats collected from 12 autologous and 18 allogeneic HSCT patients' donors were investigated by PCR before transplantation. After transplantation, blood and sera collected at fixed time intervals were screened by two PCR methods and serological assays. Screening results first time assessed a toxoplasmosis incidence level as 25% in autologous HSCT patients and increased incidence level in allogeneic HSCT patients to 22%. Importantly, buffy coat PCR was first time performed before transplantation, to determine the risk of toxoplasmosis. Buffy coat PCR results showed that four patients were at high risk of toxoplasmosis before transplantation. After transplantation, these patients experienced toxoplasmosis. In conclusion, for the determination of patients at risk of toxoplasmosis, clinicians should consider buffy coat PCR in combination with serology before transplantation. After transplantation, PCR screening can be initiated in high risk patients upon clinical suspicion.

Aim To examine performance in the UK National External Quality Assessment Scheme (UKNEQAS) for toxoplasma serology for evidence of discrepant results as compared with the predistribution and postdistribution results supplied by the toxoplasma reference laboratories.Methods Analysis of performance in the toxoplasma IgG and IgM schemes was made for the period 1994-2008 to look for trends in performance.Results For the IgG scheme, a mean of 98% of participants obtained the correct result for detection of toxoplasma-specific antibody. The most common problem was failure to detect low levels of antibody. In some cases this was the result of participants deviating from the manufacturer's instructions and using higher cut-off levels. For the IgM scheme, an average of 95% of participants obtained the correct result for toxoplasma antibody detection. The most common problem was the failure of some enzyme immunoassay kits to detect specific toxoplasma IgM antibody, which was detected by the more sensitive immunosorbent agglutination assay.Conclusions Performance standards in the UKNEQAS toxoplasma serology schemes were high. The problems encountered have highlighted the importance of detecting low levels of antibody, adhering to the kit manufacturer's instructions and selecting an appropriate assay for the clinical situation.

Serum samples from 101 stranded or bycatch cetaceans from British waters were screened for Toxoplasma gondii-specific antibodies using the Sabin Feldman Dye Test. Relatively high seropositivity was recorded in short-beaked Delphinus delphis and this study presents the first documented case of Toxoplasma in a humpback whale Megaptera novaeangliae.

Toxoplasma retinochoroiditis in pregnancy may create considerable patient anxiety and is a dilemma for the treating ophthalmologist. A case report highlighting this clinical issue is presented followed by a review of the literature. Consensus in relation to the management of toxoplasma retinochoroiditis in pregnancy is lacking and is discussed.

Toxoplasmosis is a serious and potentially life-threatening disease in liver transplant recipients while they are immunosuppressed. We report the clinical and laboratory findings related to active toxoplasma infection associated with 40 immunosuppressed liver transplant procedures that took place over a 12-month period at a major transplant unit in Izmir, Turkey. Twenty-seven (67.5%) of the 40 transplant recipients were found to be seropositive for toxoplasma infection and therefore at risk of reactivated infection. From the serological status of the donors, which was ascertained in 38 of 40 cases, we identified 3 (7.9%) of 38 transplants to be from a seropositive donor to a seronegative recipient. In 10 (26.3%) of 38 transplants, both the donor and recipient were seronegative, and this excluded toxoplasma as a risk. A comparison of real-time polymerase chain reaction (PCR) and nested PCR was undertaken in combination with a range of serological assays (the Sabin-Feldman dye test, enzyme immunoassay immunoglobulin M, and immunosorbent agglutination assay immunoglobulin M). Ethylene diamine tetraacetic acid blood samples from 3 of the 30 recipients at risk from toxoplasma were found positive by PCR, but only 1 of these was found positive in both assays. Among the 3 PCR-positive patients, immunoglobulin M and immunoglobulin G antibody levels increased in only 1 patient. Correlations between symptoms, laboratory findings, and clinical management (use of anti-toxoplasma therapy) are presented. Our findings suggest that toxoplasma presents a significant risk to our liver transplant population and that PCR is a helpful addition in identifying active infections and hence in informing clinical management decisions.

OBJECTIVE:To verify the incidence of cryptosporidiosis among Turkish elementary school students. METHODS:This study was conducted in the Department of Parasitology, Faculty of Medicine, Ege University, Turkey during a 3-month period in 2006. We assessed the fecal samples of 707 children using modified acid-fast and phenol-auramine staining followed by modified Ritchie concentration method. All Cryptosporidium species isolates were analyzed by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) to differentiate the genotypes of the isolates. After the coprological examination, 4 samples were found to be positive for Cryptosporidium species oocysts. RESULTS:In the present study, all 4 oocysts were of zoonotic origin and belonged to Cryptosporidium parvum genotype 2 indicating that in Turkey the potential sources of human cryptosporidiosis is from animals. CONCLUSION:The application of genotyping to clinical isolates of Cryptosporidium has significantly increased our knowledge and understanding of the distribution and epidemiology of this parasite. The PCR and RFLP techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional analytical DNA techniques.

ABSTRACT The LIAISON system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles. The system allows fast and precise measurement of Toxoplasma -specific immunoglobulin G (IgG) and IgM antibody levels and measurement of the IgG avidity index even at low levels of Toxoplasma -specific IgG antibodies in a single step without manual interference. Seven European centers participated in a multicenter evaluation of the LIAISON system. The sensitivity and specificity of the LIAISON system compared to the Sabin-Feldman dye test were 99.3 and 96.8%, respectively. In a comparison of the LIAISON Toxoplasma -specific IgM assay with an immunosorbent agglutination assay, the LIAISON assay had a sensitivity of 96.7% and a specificity of 95.4%. The LIAISON IgG assay showed agreements of 91, 100, and 100% with the AXSYM IgG (Abbott), VIDAS IgG (bioMérieux), and Platelia IgG (Bio-Rad) assays, respectively. The LIAISON IgM assay showed agreements of 95% with the AXSYM IgM and Platelia IgM assays, 96% with the ISAGA IgM assay (bioMérieux), and 97% with the VIDAS IgM assay. The coefficient of correlation between the LIAISON system and the VIDAS Toxoplasma -specific IgG avidity index was 0.81. By use of the Toxoplasma -specific IgG avidity index assay with specific IgM-positive samples, the diagnosis of infection with Toxoplasma gondii in early pregnancy has been improved significantly. The LIAISON avidity assay is a valuable assay for the exclusion of recently acquired infection with T. gondii (less than 4 months) in pregnant women, and it decreases significantly the necessity for follow-up testing.

Polymerase chain reaction amplification and DNA sequencing of the Toxoplasma gondii dihydropteroate synthase gene (dhps) identified 4 alleles among parasite populations from 32 cases of human toxoplasmosis. Heterologous expression and enzyme assay reveal that 3 of these alleles encode sulfadiazine (Sdz)-sensitive enzymes. The fourth, generating a highly Sdz-resistant enzyme, differs from 1 of the other 3 at only a single residue (407) of DHPS. Of interest, a fifth allele, found in a laboratory-induced Sdz-resistant line, also differs from another of these 3 drug-sensitive forms by the same single mutation that affects residue 407 of DHPS. Significantly, residues corresponding to DHPS-407 are implicated in sulfonamide resistance in other microorganisms. The human-derived allelic form encoding the Sdz-resistant enzyme was found in T. gondii associated with a fatal infection, and its presence within clinical material may have implications for sulfonamide use, particularly in cases of toxoplasmosis in which the initial response to drug treatment is poor.

The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M, IgA, IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3–12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.