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Wild lagomorphs play a key epidemiological role as reservoirs of Leishmania infantum, causative agent of the largest outbreak of human leishmaniosis in Europe to date. A large-scale survey study was conducted on wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) populations in Spanish Mediterranean ecosystems to evaluate the exposure of L. infantum and investigate potential risk factors associated with exposure to this zoonotic parasite. Between 2018 and 2021, a total of 631 wild lagomorphs (471 wild rabbits and 160 Iberian hares) were collected in Andalusia (southern Spain) and tested for antibodies against L. infantum using the indirect fluorescent antibody test (IFAT). Spleen samples from 563 of the wild lagomorphs sampled (441 wild rabbits and 122 Iberian hares) were also evaluated by real-time quantitative PCR (qPCR) for detection of Leishmania kDNA. Exposure to L. infantum (positive by IFAT and/or qPCR) was detected in 56.4 % (356/631; 95 %CI: 52.3-60.3) of the lagomorphs analyzed. Anti-Leishmania antibodies were found in 12.8 % (81/631; 95 %CI: 10.2-15.5) of the animals, and L. infantum kDNA was detected in 59.0 % (332/563; 95 %CI: 54.9-63.0) of the spleen samples tested. Phylogenetic analysis revealed high homology (99.9-100 %) between L. infantum sequences obtained and strains previously isolated from humans in Spain. While apparent seroprevalence was significantly higher in Iberian hares (19.4 %; 95 %CI: 13.3-25.5) compared to wild rabbits (10.6 %; 95 %CI: 7.9-13.4), no significant differences in prevalence were found between wild rabbits (61.0 %; 95 %CI: 56.5-65.6) and Iberian hares (51.6 %; 95 %CI: 42.8-60.5). At least one positive animal was found on 64.8 % (70/108) of the hunting grounds sampled, and a high-risk spatial cluster (P < 0.001) was also identified in central Andalusia. The multivariable analysis identified bioclimatic level (meso-Mediterranean climate) and the presence of goats on hunting grounds as risk factors potentially associated with L. infantum exposure in wild lagomorphs. This study shows high, widespread exposure, but heterogeneous distribution of L. infantum in wild lagomorph populations in Mediterranean ecosystems in southern Spain. The results point to the need to promote integrated surveillance programs for the detection of Leishmania spp. in wild lagomorphs in order to establish effective control measures against human leishmaniosis under a One Health approach., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Toxoplasmosis is a protozoan disease caused by Toxoplasma gondii. Different T. gondii confirmatory techniques, including serologic methods, are available to detect the presence of the parasite. Among serology techniques, immunochromatographic rapid testing could be a reliable alternative to serologic laboratory techniques., Objective: This study evaluated a commercial immunochromatographic test (FASTest TOXOPLASMA g) in seronegative and seropositive cats., Methods: Two indirect immunofluorescence antibody reference tests, an in-house technique, and a commercial test were used to classify 292 feline serum samples. The rapid test was evaluated in different groups of cats, including healthy seronegative cats (n = 121), seropositive cats with variable anti-Toxoplasma antibodies (n = 146), and cats with positive serologic results for other pathogens (n = 25). The sensitivity, specificity, accuracy, receiver operating characteristic curves, and kappa statistics were analyzed as performance measures., Results: Of the 292 samples, 146 were classified as T. gondii seropositive and 146 as T. gondii seronegative. Concordant results were obtained for all samples using immunofluorescence antibody tests. The diagnostic measures of this rapid test showed 98.63% sensitivity and 100% specificity, and 99.32% accuracy. The kappa statistics value was 0.986, and the area under the receiver operating characteristic curve was 0.993., Conclusions: This rapid test showed diagnostic measurements similar to those of traditional quantitative serologic methods. In situations where laboratory techniques are not available, this test, under clinical conditions, could be a useful alternative to obtain accurate results rapidly., (© 2023 American Society for Veterinary Clinical Pathology.)

Introduction: Previously, diagnosis of ocular toxoplasmosis is based on clinical symptoms and Toxoplasma serology. Checking serological indicators often cannot reflect the real intraocular situation, and may even mislead clinicians to make wrong judgments., Patient Concerns: A 38-year-old male patient visited our ophthalmology clinic with a chief complaint of decreased vision for about 5 days in his right eye., Diagnosis: Aqueous humor sample analysis found Toxoplasma DNA detectable, and Toxoplasma immunoglobulin G (IgG) and immunoglobulin M (IgM) to be positive. His serum Toxoplasma IgG was also positive (10.04 IU/mL; reference range: 0 to 7.2 IU/mL). Therefore, the final diagnose was ocular toxoplasmosis involving his right eye., Interventions: Oral prednisone 60 mg/day and azithromycin 0.25 g/day were started. Oral antibiotic treatment for toxoplasma was continued for 4 weeks, and prednisone followed by weekly stepwise tapering in steps of 10 mg/day., Outcomes: The BCVA and fundus of right eye remained stable after treatment at follow-up., Conclusions: This article reported a case of ocular Toxoplasma gondii infection diagnosis by serum and aqueous humor antibody tests. We provide some additional information on the T gondii infection diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA., Methods: A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients ( n  = 14), healthy individuals ( n  = 40), and patients with other diseases ( n  = 276)., Results: ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8-100.0] and 97.8% specificity 95% CI [95.5-99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8-100.0]) and a slightly higher specificity (99.1% 95% CI [97.3-99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar's test; p > 0.05), with a concordance of 98.2%, represented by a Cohen's kappa of 0.83 ( p < 0.001), indicating almost perfect agreement., Conclusion: ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis., Competing Interests: The authors declare there are no competing interests., (©2022 Janwan et al.)

Objective: To report on a unique combination of cerebral toxoplasmosis and ocular toxoplasmosis in an HIV-positive patient in Slovakia., Methods: A 35-year-old heterosexual patient who presented with headache and major seizures underwent computed tomography (CT) and magnetic resonance imaging (MRI). Based on clinical findings, serological tests for toxoplasmosis were performed on serum and ocular fluid specimens. PCR was also used to detect Toxoplasma gondii and cytomegalovirus DNA. Goldmann and Witmer coefficient calculation was applied to demonstrate the synthesis of intraocular IgG antibodies., Results: CT and MRI revealed cystic lesions suspected of metastasis in the occipital and temporal regions, and we searched for the primary tumor. After vision loss in the left eye, which rapidly progressed to complete blindness, an eye examination detected macular edema. Anti-edema treatment was initiated. HIV positivity with a very low CD4 T-cell count (20/&#956;L) was found, and the viral load was 100 400 HIV-RNA copies/ml. The serum was positive for anti-Toxoplasma IgG antibodies (&gt; 200 IU/mL), IgM negative, and IgA borderline. As toxoplasmic encephalitis and retinitis were suspected, antitoxoplasmic therapy with pyrimethamine, spiramycin, and folinic acid was started. The ophthalmologist considered cytomegalovirus retinitis, which was not confirmed by serology or PCR. In contrast, the presence of IgG antibodies in ocular fluid and serum with the calculation of the Goldmann-Witmer coefficient (GW = 32) as well as PCR DNA positivity pointed to Toxoplasma gondii as the etiological agent. Follow-up MRI scan confirmed regression of the pathological lesions, neurological deficit also improved, CD4 T-lymphocytes increased above 200/&#956;L, but blindness of the left eye persisted., Conclusion: CT and MRI scans offered no clue as to Toxoplasma etiology of the brain and eye involvement in an HIV-positive patient, which was only confirmed by laboratory tests. Due to the delay in the diagnosis of toxoplasmosis, time from the epileptic seizure to treatment initiation was 16 days, which assumedly caused irreversible blindness in the patient.

Toxoplasmosis is one of the most globally prevalent zoonotic infection caused by an obligate, intracellular parasite called Toxoplasma gondii . Toxoplasmosis actively triggers an acute immune response and inflammatory reactions, which causes serious pathological changes in various tissues in the human body, and more evidently localizes in different nervous tissues of various body organs. The YKL-40 is a glycoprotein secreted by numerous cell types in different patterns associated with various pathological processes such as inflammatory reactions, tissue remodeling, and fibrosis, and is a disease-specific biomarker of neuroinflammation. Therefore, this study aimed to determine whether the YKL-40 is markedly increased in toxoplasmosis or not and whether its level is different between the acute and chronic phases of the infection to determine if it can be used as a clinically useful biomarker in the diagnosis, and determination of disease severity and follow-up of toxoplasmosis. Accordingly, a total of 80 serum samples were collected from previously diagnosed female patients of different ages with toxoplasmosis. In addition, serum samples of 10 healthy females were used as the control. Patients were first divided into two groups (30 patients with acute infection, and 50 patients with chronic infection) depending on the results of detection of specific anti- Toxoplasma IgM and IgG by enzyme-linked immunosorbent assay (ELISA). The level of YKL-40 was then measured in the patients' serum by ELISA. The statistical analysis of data clearly disclosed very highly significant differences (P < 0.001) between the level of YKL-40 in the acute infection group and healthy controls, chronic infection group and healthy controls, and between the groups with acute and chronic infections. These findings led to conclude that YKL-40 classify as a unique and sophisticated biomarker in the diagnosis of toxoplasmosis where it can vitally be used to detect the stage of the disease, whether acute or chronic, besides its ability to detect the infection., Competing Interests: The authors do not have any conflicts of interest to declare.

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi uses various mechanisms of infection to access humans. Since 1967, food contaminated with metacyclic trypomastigotes has triggered several outbreaks of acute infection of Chagas disease by oral transmission. Follow-up studies to assess the effectiveness of anti-parasitic treatment of oral outbreaks are rather scarce. Here, we report a 10-year laboratory follow-up using parasitological, serological, and molecular tests of 106 individuals infected in 2007 of the largest known outbreak of orally transmitted Chagas disease, which occurred in Caracas city, Venezuela. Before treatment (2007), specific IgA, IgM and IgG, were found in 71% (75/106), 90% (95/106) and 100% (106/106), respectively, in addition to 21% (9/43) parasitemia, Complement Mediated Lysis (CML) in 98% (104/106) and 79% (34/43) parasitic DNA for PCR. Blood culture detected parasitemia up to 18 months post-treatment in 6% (6/106) of the patients. In 2017, the original number of cases in the follow-up decreased by 46% and due to the country's economic situation, not all the trials could be carried out in the entire population. During follow-up, IgA and IgM disappeared promptly, with IgM persisting in 19% (20/104) of the patients three years after treatment. The anti-T. cruzi IgG remained positive 10 years later in 41% (20/49) of the individuals evaluated. CML remained positive seven years later in 79% (65/82) of the cases. PCR positive cases decreased after treatment but progressively recovered, being positive in 69% (32/46) of the individuals evaluated in 2017. The group of children (under 18 years of age) showed the highest PCR positivity with 76% (26/34) of the cases, but their parasitic load tended to diminish, while in adults the parasitic load regained their initial values. The simultaneous evaluation of serological tests and PCR of the patients allowed us to separate patients among responders and non-responders to the anti-parasitic treatment, and this information prompted us to apply a second anti-parasitic treatment in the group of non-responders. In this population not subjected to the like lihood of re-infection, adult patients were more likely to be non-responders when compared to children. These results suggest that rigorous laboratory follow-up with T. cruzi infectious biomarkers is essential to detect cases of parasite persistence., (Copyright © 2021. Published by Elsevier B.V.)

Background: Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar., Methods: Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections., Results: Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%)., Conclusions: Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure., (© 2021. The Author(s).)

Toxoplasma gondii is a zoonotic cosmopolitan protozoan that causes a high mortality rate among zoo mammals such as New World primates, meerkats, marsupials and Pallas' cat. It has been recently reported in Chile, mainly among wild populations, but also as the cause of death of a kangaroo and a mara. However, there has not been a T. gondii report at a Zoo population level in Chile in the last 35 years. The aim of the study was to estimate the seroprevalence and risk factors associated with T. gondii infection in mammals housed in a zoo located in the Metropolitan Region of Chile between 2011 and 2018. In this study, we analyzed 350 samples, from 324 animals, belonging to 57 species of carnivores, non-human primates, macropodids, ungulates and rodents to detect the presence of anti-T. gondii antibodies. Additionally, 20 animals were longitudinally sampled to evaluate intra-zoo infection. Using a commercial indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) test, we detected T. gondii antibodies in 72 (22.2 %) samples. The overall seroprevalence estimates were 48.4 % in felines, 22.9 % in non-feline carnivores, 21.1 % in ungulates and 15.0 % in non-human primates. There were no positive samples from rodents or marsupials. Of animals sampled longitudinally, only a culpeo fox (Lycalopex cualpaeus) became seropositive along the study indicating exposition inside the facility. T. gondii seroprevalence differed significantly in taxonomic groups (p = 0.003), felines are statistically different from non-feline carnivores (NFC) (p = 0.040), ungulate (p = 0.027) and non-human primates (NHP) (p = 0.009). Annual prevalence comparison was performed showing no statistical difference (p = 0.941). A multivariable logistic regression was performed to ascertain the effect of taxonomic groups, proximity to water sources, diet, sex and type of housing on seropositivity. Only taxonomic group was statistically significant, indicating that NFC (OR = 0.35; 95 % CI = 0.15 - 0.83; p = 0.017), ungulates (OR = 0.30; 95 % CI = 0.13 - 0.69; p = 0.005), and NHP (OR = 0.25; 95 % CI = 0.09 - 0.72; p = 0.010) have lower risk of positivity to T. gondii compared to felines. Additionally, a black-faced spider monkey (Ateles chamek) and a siamang (Symphalangus syndactylus) were seropositive, being the first description of T. gondii infection in these species worldwide. As seen in previous studies, the widespread presence and exposure of T. gondii in zoo mammals was confirmed, and there may be contact with the agent and transmission within the zoo, which was confirmed by one animal became seropositive over the time. This fact could be a health problem for animals susceptible to fatal toxoplasmosis., (Copyright © 2021. Published by Elsevier B.V.)

Purpose: To determine classification criteria for toxoplasmic retinitis., Design: Machine learning of cases with toxoplasmic retinitis and 4 other infectious posterior uveitides / panuveitides., Methods: Cases of infectious posterior uveitides / panuveitides were collected in an informatics-designed preliminary database, and a final database was constructed of cases achieving supermajority agreement on diagnosis, using formal consensus techniques. Cases were split into a training set and a validation set. Machine learning using multinomial logistic regression was used on the training set to determine a parsimonious set of criteria that minimized the misclassification rate among the infectious posterior uveitides / panuveitides. The resulting criteria were evaluated on the validation set., Results: Eight hundred three cases of infectious posterior uveitides / panuveitides, including 174 cases of toxoplasmic retinitis, were evaluated by machine learning. Key criteria for toxoplasmic retinitis included focal or paucifocal necrotizing retinitis and either positive polymerase chain reaction assay for Toxoplasma gondii from an intraocular specimen or the characteristic clinical picture of a round or oval retinitis lesion proximal to a hyperpigmented and/or atrophic chorioretinal scar. Overall accuracy for infectious posterior uveitides / panuveitides was 92.1% in the training set and 93.3% (95% confidence interval 88.2, 96.3) in the validation set. The misclassification rates for toxoplasmic retinitis were 8.2% in the training set and 10% in the validation set., Conclusions: The criteria for toxoplasmic retinitis had a low misclassification rate and seemed to perform sufficiently well for use in clinical and translational research., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Different immunohistochemical markers to detect amastigotes in cutaneous leishmaniasis have been proposed with variable diagnostic usefulness., Objectives: To evaluate the diagnostic usefulness of immunohistochemical amastigotes identification by specific polyclonal anti-Leishmania antibodies and CD1a expression (clone EP3622) in a series of PCR confirmed cutaneous leishmaniasis., Materials and Methods: Thirty-three skin samples corresponding to PCR confirmed cutaneous leishmaniasis were included in the study. All samples were stained with Hematoxylin-eosin and Giemsa. Moreover, immunohistochemical studies with anti-CD1a and anti-Leishmania antibodies were performed. The patients clinical features and the observed histopathological features were also recorded., Results: From the selected 33 biopsies, Leishmania spp. amastigotes were detected in 48.4% of cases with conventional Hematoxylin-eosin stain and in 57.5% of cases by Giemsa staining. In 31/33 cases, anti-CD1a allowed us to identify parasitic structures, and in 33/33 cases amastigotes were detected with anti-Leishmania antibodies. Concordance between both techniques, anti-CD1a and anti-Leishmania, was 94% [CI 95%: (79,8%-99,3%)] ; p value <0.05. The sensitivity of anti-CD1a in comparison with the PCR was 94%, with a positive predictive value of 100%. Two cases of low parasitic index were negative for CD1a immunostaining. In cases with high parasitic index, anti-CD1a stained amastigotes in superficial and deep dermis. Only a few cases were originally diagnosed with the available histological techniques, needing PCR for Leishmania spp., Conclusions: Anti-CD1a antibody seems to be a useful technique to identify amastigotes when PCR and anti-Leishmania antibodies are not available. The sensitivity to detect amastigotes is increased when the CD1a immunostaining is added to the classical Haematoxylin - eosin and Giemsa staining.

The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease., (© 2020 John Wiley & Sons Ltd.)

Neospora caninum is a protozoan that can cause reproductive problems in several animal species. Although N. caninum infection has been reported in swine, the pathogenesis and clinical signs are not fully known in this species. The objective of this work was to evaluate the effect of experimental infection with tachyzoites of the N. caninum strain Nc1 in swine matrices at different stages of gestation. For that purpose, 12 gilts, seronegative for N. caninum and T. gondii, were selected and allocated into four groups of three animals each. Animals in group A were not inoculated (control) and animals in groups B, C, and D were inoculated intravenously with of 2.9 × 10 7 tachyzoites, 30 days before conception, and at 45 and 90 days of gestation, respectively. Temperature, heart rate, blood, saliva, and vaginal mucus samples from the animals were collected periodically until the time of delivery for the investigation of IgG and IgM antibodies against N. caninum using IFAT and PCR to detect the parasite DNA. All gilts sero-converted from 5 and 7 DPI (days postinoculation) to IgM and IgG, respectively. Two gilts showed hypothermia on the 5th and 7th DPI, and five inoculated animals had leukocytosis on the 7th DPI. It was possible to detect DNA of N. caninum in samples of saliva (33/84), vaginal mucus (17/84), and blood (2/84). Based on serology (IgM) and PCR, three animals in group B showed evidence of reappearance of the infection during pregnancy. It is concluded that N. caninum can cause clinical signs in infected swine females, in addition to indicating saliva as a suitable diagnostic biological material for the detection of N. caninum DNA in this animal species.

Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-1 19 , MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria., Competing Interests: PJ, CSm, and NA are employed by Mabtech AB, Sweden. Several of the reagents and the FluoroSpot reader system used in this study are produced by Mabtech. Mabtech has no influence on the content of this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jahnmatz, Sundling, Yman, Widman, Asghar, Sondén, Stenström, Smedman, Ndungu, Ahlborg and Färnert.)

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7&#95;0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7&#95;0801000 (G 93 -L 494 , PF3D7&#95;0801000N) and generated specific immune sera. We observed that PF3D7&#95;0801000 localizes in merozoites, and antibodies against PF3D7&#95;0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7&#95;0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7&#95;0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7&#95;0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Test kit for detection of IgG-antibodies to individual antigens of Toxoplasma gondii by immune blotting («Western blot» format) has been developed. Laboratory testing with first international WHO standard «Anti-toxoplasma serum (IgG), human, Lyophilized, 20 IU / ampoule» (NIBSC, Great Britain) demonstrated the analytical sensitivity of the new kit equal to 10 IU / ml. Study of the diagnostic efficiency of the new kit showed its high sensitivity, equal to 98.51 - 100%, and high specificity, equal to 99.5 - 100%. New test kit is intended for confirmatory testing in laboratory diagnostics of toxoplasmosis., Competing Interests: The authors declare no conflict of interest.

Equine protozoal myeloencephalitis (EPM) is an important neurologic disease of horses in the American continent caused by Sarcocystis neurona and Neospora hughesi infection. This study describes the pathological, immunohistochemical, and molecular findings of fatal cases of EPM in southern Brazil. A review was performed on a total of 13 cases compatible with EPM, which were diagnosed by postmortem examination in the period of 2010-2017. Epidemiological information was obtained from necropsy reports. Gross and histological lesions were characterized, and cases were subjected to immunohistochemistry anti-Sarcocystis neurona, Toxoplasma gondii, and Neospora spp. Molecular search was performed using ITS-1 gene PCRs. Microscopic lesions were multifocal in all cases, and more frequently observed in the spinal cord segments and in the rhombencephalon. Intralesional protozoans were histologically detected in five horses, while a positive immunostaining for S. neurona was observed in eleven cases (11/13). Through molecular techniques, six positive cases for the ITS-1 gene were detected, and obtained sequences presented highest similarity with S. neurona. EPM due to S. neurona infection represents an important neurologic disease of horses in Brazil and this disease should be considered as a main differential diagnosis in horses presenting neurologic signs.

Aims: We evaluate whether the serum and aqueous humour (AH) level of IgG anti-Hsp70.1 antibodies improved the biological diagnosis of ocular toxoplasmosis., Methods and Results: In this prospective cross-sectional and multicentre study, serum and AH were collected at the time of active uveitis. Anti-Hsp70.1-antibody levels were determined by ELISA. Patients with confirmed (Group A1, n = 21) or suspected ocular toxoplasmosis (group A2, n = 30) were enrolled, as well as a control group of patients with cataract (group B, n = 42). Serum IgG anti-Hsp70.1 antibody levels were not significantly different within the group of uveitis patients (A1, n = 21 vs A2, n = 30, P = .8) and were significantly associated with the affected retinal zone (P = .006) and with the size of the retinal lesion (P = .03). Serum anti-Hsp70.1 antibody level was positive in 10 out of the 18 patients of group A2. Significant anti-Hsp-70.1 antibody level in AH was reported in only three patients (3 eyes) with confirmed ocular toxoplasmosis., Conclusion: While the level of IgG anti-Hsp-70.1 antibody in AH did not improve the laboratory diagnosis of ocular toxoplasmosis, its level in serum was of major significance for retinal damage diagnosis., (© 2020 John Wiley & Sons Ltd.)

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide zoonotic disease, which affects most warm-blooded species. Besides the zoonotic relevance, toxoplasmosis is one of the major causes of reproductive disorders in small ruminants. A cross-sectional study was carried out to determine the seroprevalence, spatial distribution and risk factors associated with T. gondii infection in sheep and goats in southern Spain. During 2015-2017, a total of 1,943 small ruminants (998 sheep and 945 goats) from 127 flocks were tested for antibodies against T. gondii using the modified agglutination test (MAT, cut-off 1:25). Antibodies against T. gondii were detected in 464 of the 998 sheep (46.5 %; CI95 %: 43.4-49.6%) and 362 of the 945 goats (38.3 %; CI95 %: 35.2-41.4%) tested. The farm prevalence was 98.4 % (CI95 %: 95.4-100%) for sheep and 93.7 % (CI95 %: 87.6-99.7%) for goats. The generalized estimating equation analysis showed that presence of cats and existence of previous reproductive disorders were risk factors potentially associated with T. gondii seropositivity in small ruminants. Two statistically significant spatial clusters (P < 0.001) were identified. The seroprevalence observed in the present study indicates a widespread exposure to T. gondii in sheep and goats in southern Spain, which might have important implications for animal and public health. Management measures should be implemented in small ruminant farms in this region in order to reduce the risk of T. gondii infections, particularly in those areas identified in the spatial analysis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the most common parasitic zoonosis worldwide in multiples species of mammals and birds. Although free-range chickens may play a role as an important reservoir for T. gondii, there is no reliable and commercially available diagnostic test for this disease in chickens. In this study, we aimed to develop a novel Luciferase-linked Antibody Capture Assay (LACA) for the serodiagnosis of Toxoplasma infection in chickens. Recombinant nanoluciferase fused-T. gondii dense granule antigen 8 (rNluc-GRA8) was produced and applied to LACA assay as a diagnostic antigen. GRA8-LACA was tested with the sera from uninfected and experimentally infected chickens with T. gondii and other parasitic pathogens and showed unexpectedly high sensitivity (90.5%) and specificity (95.4%). Interestingly, E. coli lysate expressing rNluc-GRA8 could be applied in GRA8-LACA with 85.7% sensitivity and an increased specificity (96.9%) that gave better diagnostic performance compared to conventional ELISA. We applied our diagnostic system to examine 267 free-range chicken sera collected from 12 farms and 100 closed-house broiler chicken sera from local poultry abattoirs. The overall seroprevalence of toxoplasmosis in free-range chickens was 10.9% (95% CI: 10.6%-11.1%), while no positive case was found in broiler chickens. GRA8-LACA could be a useful diagnostic technique for T. gondii infection in chickens. The detection of T. gondii seropositive chickens in this study warns a potential risk of Toxoplasma transmission by the consumption of raw or undercooked chicken meat., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chagas is a neglected disease endemic in Latin America. Vector transmission control had been aggressively performed. Recent entomological surveillance in Brazil has revealed natural infection rates ranging from 0.40% to 0.52%. Although serological surveys are complex to develop, they are important for disease control. In this study, we validated the use of saliva in ELISA commercial kits with a cohort of 100 patients with Chagas disease followed at Hospital das Clinicas in São Paulo, Brazil, and 50 healthy controls. Five ELISA kits for detecting antibodies against Trypanosoma cruzi were tested. The best discrimination between Chagas patients and controls was observed with the Wiener kit, which yielded a sensitivity of 97% and a specificity of 100%. Our findings reveal that the use of saliva may be an alternative to large-scale screening surveys in detecting T. cruzi antibodies; it is a noninvasive sample collection method potentially key to large-scale screening in children.

Introduction and Objective: The problem of occupational biohazards is very important, especially in the field of agriculture and in human and veterinary medicine. The aim of the study was to determine the potential sources of infection in veterinary professionals with selected zoonotic agents, including: Toxoplasma gondii , Giardia duodenalis , Leptospira spp., Cryptosporidium spp. and Coxiella burnetii ., Material and Methods: A total of 50 air samples from barns, piggeries and veterinary surgeries were examined for the presence of Leptospira spp. and C. burnetii DNA. Serum samples of 86 pigs and 80 cows were tested for the presence of antibodies to Leptospira spp. and to phase I and II C. burnetii antigens. Serum of 70 cats were tested for the presence of antibodies to T. gondii and 65 samples of cat faeces for the presence of T. gondii oocysts. The presence of G. duodenalis and Cryptosporidium spp. were examined in 50 of dog faeces and 50 of bovine faeces samples., Results: DNA of Leptospira spp. was detected in 2 air samples from the piggeries (4%). C. burnetii DNA was not found in any sample. Anti- Leptospira spp. antibodies were detected in 51 (59.3%) of examined pigs. Neither anti- Leptospira spp. nor anti- C. burnetii antibodies were found among samples of bovine serum. Anti- T. gondii antibodies was found in 52 cat serum samples (74.3%). Among samples of cat faeces, no T. gondii oocysts were detected. In one sample of cattle stool (2%), G. duodenalis was detected and in another (2%) - Cryptosporidium spp. G. duodenalis was detected in 7 samples (14%) and Cryptosporidium spp. in 2 samples (2%) of dog faeces., Conclusions: The results of this study demonstrate the potential risk of infection with Leptospira spp. in veterinarians working with pigs. Veterinarians could be also be at risk of infection with T. gondii and G. duodenalis .

The aim of the present study was to investigate the prevalence and genotyping of Toxoplasma gondii in Iranian human immunodeficiency virus (HIV)-positive patients using multilocus-nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP). A total of 102 serum samples obtained from infected patients were collected from the laboratory centres in northern Iran. Anti-T. gondii antibodies and deoxyribonucleic acid (DNA) detection were accomplished by an enzyme-linked immunosorbent assay and PCR. The Mn-PCR-RFLP method was used for the genotyping of T. gondii. Overall, 68.6% (70/102) and 11.7% (12/102) of the individuals were tested positive for anti-T. gondii immunoglobulin G and T. gondii DNA, respectively. Complete genotyping was performed on 10/12 (83.3%) PCR-positive samples. Accordingly, the samples were classified as genotype #1 (type II clonal; n = 3, 30%), genotype #2 (type III clonal; n = 2, 20%), genotype #10 (type I clonal; n = 2, 20%), genotype #27 (type I variant; n = 1, 10%), genotype #35 (type I variant; n = 1, 10%) and genotype #48 (type III variant; n = 1, 10%). The results were indicative of the high frequency of the type I and type I variant of T. gondii strains in HIV-positive patients in northern Iran. Given the high prevalence of T. gondii and frequency of pathogenic types (pathogen in laboratory mice) in the patients, special measures should be taken to prevent the possible increased incidence of encephalitis by T. gondii.

Toxoplasma gondii (T gondii) infection has been associated with protection against allergy and autoimmune diseases. We investigated the effects of T gondii infection on cytokine and antibody responses in atopic and nonatopic Brazilian subjects. We have measured in whole-blood cultures, Th1 (IFN-γ and IL-12), Th2 (IL-5) and regulatory cytokine IL-10 in blood cells unstimulated and stimulated with pokeweed mitogen or T gondii soluble tachyzoites antigen (STAg) or Dermatophagoides pteronyssinus antigen. A significant negative association was found between high levels of anti-dust mite IgE and T gondii seropositivity (OR = 0.46; 95%CI = 0.25-0.85). STAg stimulation induced a mixed profile of Th1 and Th2 cytokines (IFN-γ, IL-12 and IL-5) in Tg-positive atopic individuals compared with Tg-negative atopic individuals (P < .0001, P = .033 and P = .003, respectively). In contrast, IL-10 production was not different between these groups. No association was found between T gondii infection and asthma. We hypothesized that the protective effect on atopy might be related to the strong Th1 immune response to T gondii found on the seropositive subjects. From our knowledge, this is the first study to investigate the association between atopy and T gondii infection in Brazilian subjects, analysing the cellular immune responses., (© 2019 John Wiley & Sons Ltd.)

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tick-borne diseases (TBD) occur in many temperate countries and are economically important in most tropical and subtropical areas, affecting dairy and beef cattle, as well as small ruminants. Four major tick-borne diseases have been detected in eastern and southern Africa: East Coast fever (ECF) caused by Theileria parva, Theiler 1904, anaplasmosis caused by either Anaplasma marginale, Theiler 1910, Anaplasma centrale, Theiler 1911, or Anaplasma ovis, Bevan 1912, babesiosis caused by Babesia bovis, Babes 1988 and Babesia bigemina, Smith & Kilborne 1893, and heartwater caused by Ehrlichia ruminantium Cowdry 1925. A cross-sectional survey was undertaken to determine the antibody prevalence of these TBDs and to identify the risk factors for TBD infections in the Union of the Comoros. In 2016 and 2017, 903 individual animal serum samples were collected from 429 separate farms, where the farmers answered individual questionnaires. The antibody prevalence of anaplasmosis, babesiosis (B. bigemina) and heartwater was determined by enzyme-linked immunosorbent assays (ELISA) and the antibody prevalence of ECF was assessed using an immunofluorescence antibody test (IFAT). The relationship between TBD seropositivity and livestock-related variables was assessed by multivariate analyses with standard logistic regression models. The results showed that these four TBDs were present in the Union of the Comoros with a global antibody prevalence of 15% (95% CI [12.7%; 17.3%]) for anaplasmosis, 9.2% (95% CI [6.5%, 11.9%]) for B. bigemina babesiosis, 5.3% (95% CI [3.2%, 7.4%]) for ECF and 4.6% (95% CI [3.2%, 6%]) for heartwater. We compared these findings with the abundance and distribution of several tick species known to be TBD vectors and we found a significant correlation between Rhipicephalus appendiculatus and ECF, and between Amblyomma variegatum and heartwater. We also found that two major variables were significantly correlated with B. bigemina antibody prevalence ("island" and "breeding area"), four variables were significantly correlated with anaplasmosis antibody seroprevalence ("island", "number of cattle per farmer", "number of farmers per village" and "breeding area"), two were significantly correlated with ECF antibody prevalence ("number of farmers in village" and "presence of ticks"), and three were significantly correlated with heartwater ("island", "number of cattle per farmer" and "number of farmers in the village"). Our findings confirmed livestock exposure to the four targeted TBDs of major concern for livestock development. Consequently, raising farmers' awareness and setting up a period of quarantine should be considered a priority., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Introduction: Vaccines are well-established public health interventions with major impact on the prevalence of infectious diseases, but outbreaks are occurring frequently due to primary and secondary failures, despite high coverage. Surveillance of efficacy and duration of induced immunity is a difficult task as it requires invasive blood sampling in children and teenagers. Saliva can be an acceptable alternative source of IgG to assess vaccine efficacy and toxoplasmosis incidence. We investigated IgG response for measles, mumps, rubella, and T. gondii in saliva samples of vaccinated young people., Methods: Saliva was collected from 249 public schools students from São Paulo, Brazil, aged 7 to 13 years old, during an interactive exhibition on hygiene. We used S. aureus protein A solid phase capture assay for IgG reactive to biotinylated purified proteins. Paired saliva and serum (47) were tested from young adults with serum evidence of T. gondii infection and from negative children less than 12 month old for standardization. Reproducibility was greater than 98% and sensitivity and specificity of the saliva assays were greater than 95%, as well as the concordance of paired saliva and serum samples., Results: Saliva from high school students showed a prevalence of 8.5% (95% CI: 5.0-11.9%) for anti T. gondii IgG; 96.8% (94.6-99%) of anti-measles IgG; 59.1% (53-65%) of anti-rubella IgG, and 57.5% (51.3-63.6%) of anti-mumps IgG., Discussion: The prevalence of antibodies against mumps and rubella after 6-8 years of vaccination was lower than against measles among students. The findings of this study demonstrate the feasibility of saliva sampling for follow-up of vaccine immune status in teenagers. This useful approach allows for IgG detection for vaccine control or epidemiological studies., (Copyright © 2019 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Toxoplasmosis is a common and serious parasitic infection caused by the ubiquitous obligatory intracellular protozoan organism, Toxoplasma gondii. Although infection with T. gondii is usually asymptomatic in healthy individuals, it can lead to severe pathological effects in congenital cases and immunocompromised patients. This study aimed to determine the seroprevalence of T. gondii and its predictors among pregnant women seeking prenatal and medical care at the general maternal and child health facility in Dhamar district of Dhamar governorate, Yemen., Methods: A total of 420 pregnant women were randomly selected for this cross-sectional study. Participants were screened for anti-T. gondii antibodies (i.e. immunoglobulin M; IgM and immunoglobulin G; IgG) using electrochemiluminescence immunoassay. Demographic, socioeconomic, obstetric and behavioural data were collected using a pretested questionnaire via face-to-face interview. Univariate and multivariate analyses were used to identify the independent predictors of T. gondii seroprevalence., Results: The overall seroprevalence of anti-T. gondii antibodies (IgG and/or IgM) among the participants was 21.2% (89/420; 95% CI = 17.3-25.1). Anti-T. gondii IgG antibodies were detected in 20.0% (84/420) of the women of which 12.9% (54/420) were positive for only IgG and 7.1% (30/420) were positive for both IgG and IgM antibodies. Moreover, 5 women (1.2%) were reactive only for IgM antibodies. Significant associations between T. gondii seroprevalence and history of spontaneous abortion (P <  0.001), raw vegetables consumption (P = 0.036), and presence of cats in household (P = 0.049) were reported. Multivariate analysis confirmed that history of spontaneous abortion (AOR = 4.04; 95% CI = [2.46, 6.63]) and presence of cats in household (AOR = 1.77; 95% CI = [1.02, 3.07]) are significant predictors of T. gondii seroprevalence among the studied participants., Conclusion: The study found a high seroprevalence (21.2%) of T. gondii infection during pregnancy in Dhamar district, which is significantly associated with adverse pregnancy outcomes. The provision of adequate maternal healthcare and health education pertaining to the prevention of T. gondii infection is therefore imperative to curtail the prevalence of infection among the studied population.

The diagnosis of abortion in livestock is difficult. In this paper, Neospora caninumassociated abortion in cattle and buffaloes in India is reported for the first time. A total of 184 animals (cattle, n = 133; water buffaloes, n = 51) were randomly selected for seroprevalence studies from 28 farms with a history of abortion. Antibodies to N. caninum were detected using a commercial competitive enzymelinked immunosorbent assay (c-ELISA); the percentage seropositivity was 10.5% for cattle and 21.6% for buffaloes. Risk factors such as species, i.e. buffaloes in comparison to cattle (odds ratio [OR] = 2.33, calculated probability [p] = 0.05), disposal of foetus and foetal membranes by throwing them into the field, in comparison with burning/burial (OR = 2.63, p = 0.03), and origin of the animal, i.e. purchased from outside in comparison to born on the farm (OR = 4.69, p = 0.002), were significantly associated with N. caninum seropositivity in univariate logistic regression. In multivariate analysis, only two risk factors, animal purchased from outside (OR = 6.15, p = 0.001) and buffaloes (OR = 3.20, p = 0.01), were significantly associated with Neospora seropositivity at a p-value < 0.05. Histopathological examination of aborted foetal tissues (cattle, n = 13; buffaloes, n = 8) revealed N. caninum tachyzoites in the heart and liver of three foetuses, and the diagnosis was confirmed by positive immunoreactivity to anti-N. caninum polyclonal antibody in the placenta of one foetus. To the authors' knowledge, this is the first report of confirmed neosporosis abortion in cattle and buffaloes in India.

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

School-age children are a social group in which blood collection for laboratory testing can be perceived as an invasive procedure, with low acceptance and tolerance of stakeholders. This problem could be circumvented by replacing serum samples with saliva. For this purpose, and to make the collection of saliva samples playful and instructive for children, educational activities on hygiene and toxoplasmosis transmission and prevention were performed using toys and audiovisual tools. The target audience consisted of 7-10 year-old children from low-income families who attended public schools in the city of São Paulo. Saliva samples were used in a previously described in-house Enzyme-Linked Immunosorbent Assays (ELISA) to detect anti- Toxoplasma gondii IgG antibodies and establish the immunological status of each of the participants. One year later, children's memory and fixation of concepts regarding hygiene habits, as well as transmission and prevention of toxoplasmosis were tested in the same schools, by means of a questionnaire application, using students who did not participate in the first intervention as controls. The prevalence of positive anti- T. gondii IgG among students was 50% (82/164). One year later, 45 children had more knowledge on toxoplasmosis (28/45 vs 29/147) and they drew the cat's involvement in the transmission of toxoplasmosis more often than controls (28/45 vs 29/147). Sorted according to the presence of specific IgG in saliva, recovered positive students presented worse memory of the above cited knowledge as did saliva-negative IgG students, but both groups had isolated higher frequency of fixed knowledge than non-intervened students. Our data show that there is a high prevalence of T. gondii infection in school-children from low-income areas; saliva is an alternative to blood for anti- T. gondii IgG detection; and a one-day educational intervention in school-children was effective in promoting knowledge fixation on hygiene and toxoplasmosis transmission and prevention after one year.

Background: The ability to report vaccine-induced IgG responses in terms of µg/mL, as opposed arbitrary units (AU), enables a more informed interpretation of the magnitude of the immune response, and better comparison between vaccines targeting different antigens. However, these interpretations rely on the accuracy of the methodology, which is used to generate ELISA data in µg/mL. In a previous clinical trial of a vaccine targeting the apical membrane antigen 1 (AMA1) from Plasmodium falciparum, three laboratories (Oxford, NIH and WRAIR) reported ELISA data in µg/mL that were correlated but not concordant. This current study sought to harmonize the methodology used to generate a conversion factor (CF) for ELISA analysis of human anti-AMA1 IgG responses across the three laboratories., Methods: Purified IgG was distributed to the three laboratories and, following a set protocol provided by NIH, AMA1-specific human IgG was affinity purified. A new "harmonized CF" was generated by each laboratory using their in-house ELISA, and the original clinical trial ELISA data were re-analysed accordingly., Results: Statistical analysis showed that the data remained highly correlated across all three laboratories, although only Oxford and NIH were able to harmonize their CF for ELISA and generate concordant data., Conclusions: This study enabled two out of the three laboratories to harmonize their µg/mL readouts for the human anti-AMA1 IgG ELISA, but results reported from WRAIR are ~ twofold higher. Given the need to validate such information for each species and antigen of interest, it is important to bear in mind these likely differences when interpreting µg/mL ELISA data in the future.

Purpose: The aim of this study was to provide a retrospective analysis of the presentation, demographics, and treatment regimens for ocular toxoplasmosis at a large tertiary referral uveitis center., Design: Retrospective cohort study., Participants: A total of 48 patients with ocular toxoplasmosis who presented to Sydney Eye Hospital participated in this study., Methods: This is a retrospective review of patient files who presented to Sydney Eye Hospital between 2007 and 2016 with clinical features consistent with ocular toxoplasmosis. Baseline risk factors and treatment details were recorded and analyzed. Main outcome measures were visual acuity and relapse rate compared with other studies in ocular toxoplasmosis., Results: The median age was 35.5 (interquartile range 21-50) with 30 (60%) patients having no previous symptomatic episodes or evidence of chorioretinal scarring. Visual acuity at presentation was 0.51 or 6/19 (SE 0.096) and at follow-up 0.31 or 6/12 (SE 0.094). Nine patients experienced a recurrence during the period of observation with median time to recurrence 2.2 years (SE 0.45) and the relapse rate was 0.09/person-years. Location of lesion was predominantly within the vascular arcades (n = 44) with macular involvement in 9 patients. Most patients received clindamycin therapy (n = 34) with pyrimethamine and sulfadiazine was used for those with macula involvement., Conclusions: Patients with ocular toxoplasmosis had fewer recurrences compared with other published series and had better visual recovery. The majority of patients received clindamycin and oral prednisolone which were well tolerated with pyrimethazine and sulfadiazine reserved for those with macula-involving disease.

Toxoplasmosis is a common infection whose worldwide prevalence is estimated at 30%, with large disparities across the world. Among infected subjects, the prevalence of ocular toxoplasmosis (OT) is, however, limited to about 2% in Europe and 17% in South America. In France, it is estimated that about 1 000 000 patients present either active OT or subsequent chorioretinal scars. Toxoplasma gondii is the first cause of posterior uveitis worldwide, responsible for retinochoroiditis, at times associated with anterior uveitis. To date, there is no consensus yet on how to diagnose OT, which is often based only on clinical presentation. Nevertheless, OT-associated symptoms are often atypical and misleading. Over the last 20 years, tremendous progress has been made in biological tools, enabling parasitologists to confirm the diagnosis in most suspected cases of OT. Using anterior chamber puncture, a safe and fast procedure, ophthalmologists sample aqueous humour for analysis using multiple techniques in order to reach high specificity and sensitivity in OT diagnosis. In this article, we present the different techniques available for the biological diagnosis of OT, along with their characteristics, and propose a diagnostic algorithm designed to select the best of these techniques if clinical examination is not sufficient to ascertain the diagnosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Neospora caninum is an apicomplexan parasite distributed worldwide. Although a positive association between the presence of birds and abortions in cattle associated to N. caninum has been reported, the role of the birds in the epidemiologic cycle of the parasite is unknown. To the best knowledge, no experimental studies have evaluated N. caninum in the eared dove, Zenaida auriculata. Therefore, we aimed to determine whether Z. auriculat can act as intermediate host for N. caninum. Eighteen birds were divided into four groups, G1, G2, G3, and G4 (control); G1, G2 and G3 received 2 × 10 6 tachyzoites of NC-1 strain via different routes: subcutaneous, intramuscular, and intraperitoneal, respectively. G4 composed of three birds. Serum samples were collected weekly, and one bird each from G1, G2 and G3 was euthanized on the 7th and 14th day post-inoculation (dpi). The remaining birds were euthanized after the 28th dpi. Tissues from the doves were evaluated using histopathological analysis, PCR and dog bioassay to detect the parasite. Dogs were fed with tissues from the birds and monitored for 30 days. Serum samples were collected weekly from the dogs for serological analysis, and feces samples were collected daily until the end of the experiment for coproparasitological examinations. No dove showed clinical signs of the infection; however, all of them seroconverted after the inoculation, with stronger immunological response in the G3 birds. The lung tissue of one G3 bird showed positive PCR results; it was euthanized on the 7th dpi, and an inflammatory infiltrate was observed in the lung and kidney from this dove. The dogs did not shed oocysts or seroconverted. Our results indicate that the intraperitoneal route induced infection in the doves; however, the parasite may have been eliminated by the host, and the doves may be resistant to chronic infection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Congenital TORCH (toxoplasmosis, other viruses [varicella-zoster virus, VZV, etc.], rubella, cytomegalovirus [CMV], Herpes simplex virus [HSV]) infections are major causes of prenatal, perinatal, and postnatal morbidity and mortality. Although treatment or prevention strategies are available for these pathogens, all drugs may not be safe during the pregnancy. The aim of this study is to measure the antibodies (Abs) concentration in the intravenous immunoglobulin (IVIG) preparation to evaluate the therapeutic potential for TORCH infection., Methods: We tested the only one commercial IVIG preparation from Taiwanese for the presence of Abs against Toxoplasma gondii, VZV, Epstein-Barr virus (EBV), measles, mumps, rubella, CMV, HSV type 1 (HSV-1), and HSV type 2 (HSV-2) by using enzyme-linked immunosorbent assay or chemiluminescent microparticle immunoassay., Results: In our study, the median level (range) of anti-CMV immunoglobulin G (IgG) is > 250 (All > 250) (arbitrary unit, AU)/mL, anti-EBV > 200 (All > 200) (relative unit, RU)/mL, anti-HSV > 200 (152.75 to >200) RU/mL, anti-VZV > 5000 (All > 5000) IU/L, anti-measles > 5000 (All > 5000) IU/L, anti-mumps > 200 (156.5 to > 200) RU/mL, anti-rubella 209.8 IU/mL (192.7 to 238.5), and anti-Toxoplasma is 14.05 (12.3 to 16) IU/mL. There was not any immunoglobulin M (IgM) against HSV, VZV, mumps, measles, rubella, CMV, EBV, and Toxoplasma in the "Taiwan Blood Services Foundation" IVIG preparations., Conclusion: There was high activity against T. gondii, VZV, EBV, measles, mumps, rubella, CMV, HSV-1, and HSV-2 in all IVIG batches. Further investigation is warranted to confirm the efficacy of IVIG from Taiwanese for congenital TORCH infections.

Background: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs., Methodology: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera., Results: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60-97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive., Conclusion: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL., Competing Interests: BCBH was and PM is employee of Coris BioConcept and they do not have any share in the company. Sergey Litvinov is an employee of Aptum Biologics Ltd. which is an official partner institution in the Consortium that produced this work. Aptum Biologics Ltd. has and provided to the Consortium the software for epitope prediction, and beyond this has no consultancy, employment or any other forms of financial benefits from this work, nor affiliation with whatever potential intellectual property that may arise from this particular work. All other authors have declared that no competing interests exist.

Sampling the blood compartment by an invasive procedure such as phlebotomy is the most common approach used for diagnostic purposes. However, phlebotomy has several drawbacks including pain, vasovagal reactions, and anxiety. Therefore, alternative approaches should be tested to minimize patient's discomfort. Saliva is a reasonable compartment; when obtained, it generates little or no anxiety. We setup a multiplexed serology assay for detection of Toxoplasma gondii IgG and IgM, rubella IgG, and CMV IgG, in serum, whole blood, and saliva using novel plasmonic gold (pGOLD) chips. pGOLD test results in serum, whole blood, and saliva were compared with commercial kits test results in serum. One hundred twenty serum/saliva sets (Lyon) and 28 serum/whole blood/saliva sets (Nice) from France were tested. In serum and whole blood, sensitivity and specificity of multiplex T. gondii, CMV, and rubella IgG were 100% in pGOLD when compared to commercial test results in serum. In saliva, sensitivity and specificity for T. gondii and rubella IgG were 100%, and for CMV IgG, sensitivity and specificity were 92.9% and 100%, respectively, when compared to commercial test results in serum. We were also able to detect T. gondii IgM in saliva with sensitivity and specificity of 100% and 95.4%, respectively, when compared to serum test results. Serological testing by multiplex pGOLD assay for T. gondii, rubella, and CMV in saliva is reliable and likely to be more acceptable for systematic screening of pregnant women, newborn, and immunocompromised patients.

Visceral Leishmaniasis is a public health problem caused by protozoans of the genus Leishmania. K39 serological test is commonly used in the initial investigation, with high specificity, but variable sensitivity. Amastigotes can be identified by optical microscopy, however, the differential diagnosis with cellular debris or other intracellular parasites is necessary. Recent studies have raised the possibility of using immunohistochemistry in the diagnosis of visceral leishmaniasis with labeling of amastigotes by the anti-CD1a antibody. This retrospective study was based on 38 samples from patients with visceral leishmaniasis whose diagnoses were confirmed by myelogram and/or k39 testing, aside from positive (N=13) and negative biopsies (N=25), 2 samples from patients with false positive biopsies for visceral leishmaniasis and 8 samples from patients with histoplasmosis diagnosis. The histological slides were evaluated for the presence of amastigotes and their Modified Ridley Parasitic Index. The samples were submitted to immunohistochemical reactions using the anti-CD1a antibody with MTB1 and O10 clones. Immunohistochemical reactions with MTB1 and O10 clones had low sensitivity in this study. However, all bone marrow samples were previously decalcified with nitric acid which is probably a deleterious treatment for immunohistochemical reactions in this site. Excluding these samples, we obtained 58.33% sensitivity and 100% specificity with the MTB1 clone. Despite the intermediate sensitivity, the immunohistochemistry for the CD1a marker with clone MTB1 can be useful in the differential diagnosis of visceral leishmaniasis, helping to discriminate leishmania amastigotes from other pathogens with similar morphology and cellular debris in different samples, except in bone marrow biopsies previously decalcified with nitric acid.

Background: US blood donors are tested for Trypanosoma cruzi antibodies only at their first presentation, based on studies, reviewed here, demonstrating the absence of incident infections. Reports of autochthonous human transmissions of the parasite in Texas have raised concern about the safety of one-time testing., Methods: Positive donation frequencies were evaluated among first-time blood donations from 2007 to 2015. Rates and their temporal changes were evaluated in an area of high T. cruzi infection and compared with rates elsewhere. Donors with positive results were surveyed for risk factors and relevant demographic characteristics., Results: Data from 9.1 million first-time donations were analyzed; 585 (1:15,544) were confirmed positive by radioimmunoprecipitation assay (RIPA) or concordantly positive with a second screening test/licensed assay. Seroprevalence in first-time donors in Southern California (an area of high endemicity) was 1:2,747, or 5.7-fold higher than the overall rate. Rates did not change over time nationally but showed a nonsignificant consistent downward trend in Southern California. The majority (92%) of donors who responded to a questionnaire had one or more T. cruzi endemic-area risk factors. Five donors with likely autochthonous infection were identified (2007-2013); nine additional donors had RIPA false positivity., Conclusion: T. cruzi seroprevalence among donors nationally and in an area of high enzootic infection were stable or declining. Almost all interviewed seropositive donors had known risk factors indicating likely infection years earlier while residing in T. cruzi-endemic areas. In the United States, there was no evidence of increased T. cruzi prevalence among first-time donors., (© 2018 AABB.)

Neurocysticercosis is the most common helminth infection of nervous system in humans caused by the encysted larvae of Taenia solium. It is a major cause of epilepsy in tropical areas and the most common cause of focal-onset seizures in North Indian children. Children with neurocysticercosis have pleomorphic manifestations depending on the location, number and viability of the cysts and host response. In endemic areas, neurocysticercosis should be clinically suspected in any child with recent-onset seizures, headache or focal motor deficits where there is no other suggestion of an underlying neurological disorder. Diagnosis of neurocysticercosis is essentially based on neuroimaging; visualization of a scolex is diagnostic. Management includes use of cysticidal drugs usually albendazole, which seems to be effective for lesion resolution and seizure remission, use of steroids and anti-epileptic drugs. Single lesions portend good prognosis with resolution of lesions in >60% of the cases within 6 mo and good seizure control. Prognosis is guarded in cysticercus encephalitis, racemose and extraparenchymal neurocysticercosis.

Chronic skin lesions constitute a clinical diagnostic challenge. We report the case of a patient whose facial lesion was histopathologically compatible with squamous cell carcinoma and hence programmed for Mohs surgery. However, review of the clinical and epidemiological history led to laboratory diagnosis of cutaneous leishmaniasis, treatment with miltefosine, and complete resolution of the lesion.

Purpose: Ocular toxoplasmosis, which is caused by the single-cell parasite Toxoplasma gondii, is currently the most significant cause of posterior uveitis in the world. No previous studies have described the prevalence and clinical features of ocular toxoplasmosis in the northeast of Iran. The purpose of the current study was to address this gap., Methods: In this retrospective study, the medical records of 488 uveitis patients who presented to the Khatam-al-Anbia Eye Hospital of Mashhad University of Medical Sciences, a tertiary ophthalmology center in the northeast of Iran, between January 2013 and December 2015 were evaluated. The clinical features and risk factors of 99 (20%) consecutive patients with ocular toxoplasmosis were extracted., Results: Ninety-nine including 53 (53.5%) female and 46 (46.5%) male patients with ocular toxoplasmosis were included in the analysis. Reduced vision (77%) and floaters (15.2%) were the most common presenting symptoms. The age category that was most affected by ocular toxoplasmosis was 20-40 years (range: 11-65 years) with a mean age of 27.2. All patients had retinochoroiditis, but just two had anterior uveitis. All of the extracted patients, with the exception of three patients, had unilateral involvement. None of the patients had any other medical disorders with the exception of one woman, who had diabetes. Only four recurring ocular toxoplasmosis patients were referred to the education hospital during the study. Serology data were available for just 32 patients, of which 31 (96.8%) were IgG positive, and 1 (3.2%) was IgM positive., Conclusion: Toxoplasma gondii was responsible for 20% of the patients of uveitis that presented to the largest ophthalmology center in the northeast of Iran. There is a high incidence of patients of ocular toxoplasmosis in the northeast of Iran, and it is a significant cause of uveitis and visual impairment in this area.

Objective: Toxoplasma gondii is cause of a wide variety of infections in human. The aim of this study was; to evaluate the frequency of sero-positivity of the members in a family with a positive serologic person., Methods: A case-control study with 170 enrolled units which was conducted in Iran in 2017. The units were settled into two group: I: the family members of seropositive individuals and II: the family members of seronegative individuals. The level of IgG and IgM anti-toxoplasma antibodies were evaluated by ELISA qualitative manner in both groups., Results: Frequency of individuals with positive serology was 52.9% and 34.1% in group I and II, respectively (P value = 0.01)., Conclusions: Clusters of toxoplasma infection would be an event in the family members. Therefore evaluation of the family members (especially high risk persons) of a patient may be necessary., (©The Author 2018. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

The studies were carried on raccoons from Poland, Germany and the Czech Republic. Tissue samples from raccoon hearts, lungs and brains were used for molecular examination while meat juice was collected for immunological tests. Antibodies against T. gondii were detected in six out of 44 raccoons (13.6%), while T. gondii DNA was found in 18 (40.9%). Antibodies against N. caninum were found in seven raccoons (15.9%) but no parasite DNA was observed in any sample. DNA of T. gondii was observed in raccoons of both sexes (in 42.3% of females and 38.9% of males) from all three countries. The proportion of raccoons that tested positive for DNA of T. gondii was higher in the Czech Republic (47.1%) than in Germany (33.3%), however the difference was non-significant (p = 0.7032). It seems that the raccoons appear to have been exposed to both T. gondii and N. caninum, but only T. gondii infection was confirmed. The role of raccoons as reservoir, and as possibly contributing to spread of these parasites merits further studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A major public health issue, Toxoplasma gondii infection can affect humans mainly via the consumption of animal products from certain species, including small ruminants. Therefore, a regular monitoring of the infection in ovine and caprine populations is advisable for the control of human and animal toxoplasmosis. Antibody detection in individual and bulk tank milk (BTM) may represent a valid alternative to serological analysis, in that its collection is easy and does not affect animal welfare. Many serological tools for milk analysis have already been validated for several parasites, including Apicomplexa. Thus, the aim of the present study was to obtain epidemiological data on T. gondii infection through the detection of antibodies in BTM of dairy goat herds from an important area for caprine dairy production (northern Italy). The performance of a commercial ELISA was first evaluated for analysis of caprine milk samples, using a panel of serum-milk pairs of goats naturally infected by T. gondii. The analysis of BTM confirmed the presence of anti- T. gondii antibodies in 59% of the samples. Toxoplasma gondii antibody positivity was more frequently found in goats reared on farms under extensive (64.9%) or semi-intensive systems (68.7%) in comparison with intensive farms (51.1%). Analysis of milk was a valid alternative to serological tests, being easily applied in large-scale epidemiological surveys and for continuous monitoring of T. gondii infection.

Background: Ocular toxoplasmosis (OT) is a common cause of posterior uveitis worldwide. The diagnosis of OT is based on clinical findings, but in most cases, laboratory tests are required to confirm the aetiology, especially when other diseases are suspected. The aim of this study was to evaluate which methods, between the Goldmann-Witmer coefficient (GWC) and immunoblotting (IB) with both IgG and IgA, in aqueous humour (AH) samples, can be the most sensitive to diagnose OT, in current practice, especially in the first three weeks., Methods: Retrospectively reviewed records of 87 consecutive patients who had underwent AH and serum sample, 42 patients with suspected OT and 45 patients with suspected other ocular inflammatory diseases. All samples were analysed by both GWC and IB., Results: The GWC was significant in 47.6% of patients presenting with suspected OT. The intraocular production of specific antibody anti-Toxoplasma gondii IgG and IgA was revealed by IB in 71.4% of samples. The combination of these two methods increased the sensitivity to 76.2%. Based on the interval between symptom onset and paracentesis, IB had a greater sensitivity than GWC when sample of AH was taken in the first three weeks (64.7% vs 23.5%, P=0.039), while the difference between the sensitivity of IB and GWC was less important in cases with an interval >3 weeks (76% vs 64% P=0.625)., Conclusion: IB seems to be more useful than the GWC if only one of these methods can be performed, especially during the first three weeks after symptom onset., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)