Advancing Toxoplasma gondii multiplex serology.

Toxoplasma 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. IMPORTANCE: Toxoplasma 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.

Calcium signaling mediates proliferation of the precursor cells that give rise to the ciliated left-right organizer in the zebrafish embryo.

Several of our internal organs, including heart, lungs, stomach, and spleen, develop asymmetrically along the left-right (LR) body axis. Errors in establishing LR asymmetry, or laterality, of internal organs during early embryonic development can result in birth defects. In several vertebrates-including humans, mice, frogs, and fish-cilia play a central role in establishing organ laterality. Motile cilia in a transient embryonic structure called the "left-right organizer" (LRO) generate a directional fluid flow that has been proposed to be detected by mechanosensory cilia to trigger asymmetric signaling pathways that orient the LR axis. However, the mechanisms that control the form and function of the ciliated LRO remain poorly understood. In the zebrafish embryo, precursor cells called dorsal forerunner cells (DFCs) develop into a transient ciliated structure called Kupffer's vesicle (KV) that functions as the LRO. DFCs can be visualized and tracked in the embryo, thereby providing an opportunity to investigate mechanisms that control LRO development. Previous work revealed that proliferation of DFCs via mitosis is a critical step for developing a functional KV. Here, we conducted a targeted pharmacological screen to identify mechanisms that control DFC proliferation. Small molecule inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) were found to reduce DFC mitosis. The SERCA pump is involved in regulating intracellular calcium ion (Ca2+) concentration. To visualize Ca2+ in living embryos, we generated transgenic zebrafish using the fluorescent Ca2+ biosensor GCaMP6f. Live imaging identified dynamic cytoplasmic Ca2+ transients ("flux") that occur unambiguously in DFCs. In addition, we report Ca2+ flux events that occur in the nucleus of DFCs. Nuclear Ca2+ flux occurred in DFCs that were about to undergo mitosis. We find that SERCA inhibitor treatments during DFC proliferation stages alters Ca2+ dynamics, reduces the number of ciliated cells in KV, and alters embryo laterality. Mechanistically, SERCA inhibitor treatments eliminated both cytoplasmic and nuclear Ca2+ flux events, and reduced progression of DFCs through the S/G2 phases of the cell cycle. These results identify SERCA-mediated Ca2+ signaling as a mitotic regulator of the precursor cells that give rise to the ciliated LRO.

Discordant Congenital Toxoplasmosis and Cytomegalovirus Infection in Dichorionic Diamniotic Twins.

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.

Fifteen-minute consultation: Management of the infant born to a mother with toxoplasmosis in pregnancy.

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.

Validation of Multiplex Serology for human hepatitis viruses B and C, human T-lymphotropic virus 1 and Toxoplasma gondii.

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.

Toxoplasmosis in Transplant Recipients, Europe, 2010-2014.

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.

Continuous versus intermittent endotracheal cuff pressure control for the prevention of ventilator-associated respiratory infections in Vietnam: study protocol for a randomised controlled trial.

BACKGROUND: Ventilator-associated respiratory infection (VARI) comprises ventilator-associated pneumonia (VAP) and ventilator-associated tracheobronchitis (VAT). Although their diagnostic criteria vary, together these are the most common hospital-acquired infections in intensive care units (ICUs) worldwide, responsible for a large proportion of antibiotic use within ICUs. Evidence-based strategies for the prevention of VARI in resource-limited settings are lacking. Preventing the leakage of oropharyngeal secretions into the lung using continuous endotracheal cuff pressure control is a promising strategy. The aim of this study is to investigate the efficacy of automated, continuous endotracheal cuff pressure control in preventing the development of VARI and reducing antibiotic use in ICUs in Vietnam. METHODS/DESIGN: This is an open-label randomised controlled multicentre trial. We will enrol 600 adult patients intubated for ≤ 24 h at the time of enrolment. Eligible patients will be stratified according to admission diagnosis (180 tetanus, 420 non-tetanus) and site and will be randomised in a 1:1 ratio to receive either (1) automated, continuous control of endotracheal cuff pressure or (2) intermittent measurement and control of endotracheal cuff pressure using a manual cuff pressure meter. The primary outcome is the occurrence of VARI, defined as either VAP or VAT during the ICU admission up to a maximum of 90 days after randomisation. Patients in both groups who are at risk for VARI will receive a standardised battery of investigations if their treating physician feels a new infection has occurred, the results of which will be used by an endpoint review committee, blinded to the allocated arm and independent of patient care, to determine the primary outcome. All enrolled patients will be followed for mortality and endotracheal tube cuff-related complications at 28 days and 90 days after randomisation. Other secondary outcomes include antibiotic use; days ventilated, in ICU and in hospital; inpatient mortality; costs of antibiotics in ICU; duration of ICU stay; and duration of hospital stay. DISCUSSION: This study will provide high-quality evidence concerning the use of continuous endotracheal cuff pressure control as a method to reduce VARI, antibiotic use and hospitalisation costs and to shorten stay. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02966392 . Registered on November 9, 2016. Protocol version: 2.0; issue date March 3, 2017.

East-West Divide: temperature and land cover drive spatial variation of Toxoplasma gondii infection in Eurasian otters (Lutra lutra) from England and Wales.

Toxoplasma gondii, a zoonotic parasite of global importance, infects all endothermic vertebrates, with extensive health implications. The prevalence of this parasite is seldom monitored in wildlife. Here, a semi-aquatic species, the Eurasian otter (Lutra lutra) was used as a model to assess the potential effect of climate, land cover and biotic factors on T. gondii seroprevalence in British wildlife. The Sabin-Feldman cytoplasm-modifying dye test identified T. gondii antibodies in 25·5% of blood samples from otters found dead, mainly as road kill, in England and Wales, between 2004 and 2010. Otters in the east of England were more likely to be infected with T. gondii than those in western regions. Land cover and temperature are key determinants of T. gondii infection risk, with more infection in arable areas and lower infection where temperatures are higher. The probability of T. gondii infection increased with host age, reflecting cumulative exposure with time, but there was no association between T. gondii seroprevalence and cause of host death.

Establishment of replacement International Standard 13/132 for human antibodies to Toxoplasma gondii.

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.

Isolation and characterization of Borrelia burgdorferi strains from Ixodes ricinus ticks in the southern England.

INTRODUCTION: Lyme disease is a bacterial infection caused by the spiral-shaped bacterium Borrelia burgdorferi. We investigated the presence and prevalence of Borrelia species in ticks from the southern England. METHODS: One hundred fifty-five cases (103 adult and 52 nymphal ticks) were collected from animal carcases. The midguts were removed, cultured in Barbour/Stoenner/Kelly II (BSK-II) and Barbour/ Stoenner/Kelly F (BSK-F) media and examined by IF, dark-field microscopy, and nested PCR. RESULTS: From a total 155 cultured ticks, two showed evidence of spirochetes and denoted as SO-1 and SO-2 strains. The availability of these two isolates enabled their antigenic characterization with SDS-PAGE and western blotting and comparison with two standard isolates. These studies identified six protein antigens with molecular weights of 18, 30, 39, 47, 60 and 88 kDa with particular promise for detecting specific immune responses to B. burgdorferi infection including Lyme disease. We also investigated the effect of repeated subculture on the antigenic pattern of UK isolate of B. burgdorferi. CONCLUSION: As a result of this study, antigenic differences have been seen between the UK isolates and the foreign isolates used as laboratory standards.

Associations between active school transport and physical activity, body composition, and cardiovascular fitness: a systematic review of 68 studies.

BACKGROUND: The impact of active school transport (AST) on daily physical activity (PA) levels, body composition and cardiovascular fitness remains unclear. METHODS: A systematic review was conducted to examine differences in PA, body composition and cardiovascular fitness between active and passive travelers. The Medline, PubMed, Embase, PsycInfo, and ProQuest databases were searched and 10 key informants were consulted. Quality of evidence was assessed with GRADE and with the Effective Public Health Practice Project tool for quantitative studies. RESULTS: Sixty-eight different studies met the inclusion criteria. The majority of studies found that active school travelers were more active or that AST interventions lead to increases in PA, and the quality of evidence is moderate. There is conflicting, and therefore very low quality evidence, regarding the associations between AST and body composition indicators, and between walking to/from school and cardiovascular fitness; however, all studies with relevant measures found a positive association between cycling to/from school and cardiovascular fitness; this evidence is of moderate quality. CONCLUSION: These findings suggest that AST should be promoted to increase PA levels in children and adolescents and that cycling to/ from school is associated with increased cardiovascular fitness. Intervention studies are needed to increase the quality of evidence.

Seroprevalence of Toxoplasma gondii in the Eurasian otter (Lutra lutra) in England and Wales.

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.

Determining Toxoplasma high-risk autologous and allogeneic hematopoietic stem cell transplantation patients by systematic pre-transplant PCR screening of stem cell originated buffy coat.

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.

The United Kingdom National External Quality Assessment Service for parasitology: toxoplasma serology scheme.

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.

The sero-prevalence of Toxoplasma gondii in British marine mammals.

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.

The sero-prevalence of Toxoplasma gondii in British marine mammals

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: Using current evidence to inform management.

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.

Incidence and diagnosis of active toxoplasma infection among liver transplant recipients in Western Turkey.

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.

The prevalence of cryptosporidiosis in Turkish children, and genotyping of isolates by nested polymerase chain reaction-restriction fragment length polymorphism.

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.