The Venn diagram in Fig. 1a provides a model and an explanation that addresses why some persons might have diseases to which T. gondii contributes, as suggested by our current analyses, and why other persons do not have these diseases. We hypothesize that disease occurs in the presence of the relevant susceptibility genes, parasite genotype and other innate and environmental factors such as other infections, the microbiome, or stress that influence immune responses, as shown in Fig. 1a. This reconstruction and deconvolution analysis presented herein (Fig. 1b,c) and summarized schematically in Fig. 1d unveils a plethora of pathways known in neurologic, immune and endocrine systems. These are likely to interact to cause neuropathologic diseases, depending on genetics of infected individuals (Figs 1a, 2a). Focus on neuronal stem cells reveals pathologic mechanisms in neurodevelopment and neuroplasticity. For example, cytokines mediate migration and homing of immune cells that serve parallel roles in spatial guidance of NSC in proper development and plasticity of human brain54. Cytokines stimulating CD8+ T cells to control T. gondii in brain55 may disrupt normal brain development and plasticity. A wide range of cytokines are evident in our data and interactome. Little is known about endocrine-type mechanisms in T. gondii infection56. This study provides a foundation to delineate endocrine influences and cross-talk with immune and neural systems.

Certain of our observations of specific genes/molecules, interactions, and pathways apparent in our data sets are particularly noteworthy: For example, in our genetics data (Table 1, Fig. 2), TREX1, TLR9, TIRAP Mal, ALOX12, NALP1, NFκB, and TGFβ connect to pathogen sensing, changes in lipids, and cell death and replication (Fig. 2). Characterization of phenotypes of our newly identified susceptibility genes is ongoing (Table 1): TREX1 was studied because of the similarity between the brain calcifications in congenital toxoplasmosis and in the genetic Aicardi Goutiere’s disease that could be due to mutations in TREX1 (Naranjo-Galvis et al., manuscript in preparation). TIRAP MAL (Hargrave et al., manuscript in preparation) (Table 1) was selected as a candidate gene studied individually as a downstream signaling molecule from TLR2 and 4. FOXQ (Table 1) was next chosen as a candidate gene because of a hydrocephalus association and subsequently has been shown to have a natural killer (NK) cell mediated phenotype33. TLR9 was studied next as a candidate gene involved in recognition of small fragments of DNA (Hargrave et al., manuscript in preparation). Noting this association for TLR9 in the NCCCTS (Table 1), we also have replicated the importance of TLR9 in susceptibility to ocular toxoplasmosis in a cohort in Brazil32.

The localization of these T. gondii susceptibility genes in human brain (Fig. 2a) also provides insights consistent with our clinical observations of toxoplasmosis in mice and humans who develop seizures originating in the hippocampus-temporal lobe, impaired movement and hydrocephalus4, 41. Z-scores indicated that all of the 17 genes except COL2A are downregulated in the hippocampus. Memory, spatial navigation and control of attention occur in the hippocampus, which is also the dominant niche for neural stem cells. In the choroid plexus of the lateral ventricle where cerebrospinal fluid is produced, eight susceptibility genes are up-regulated (ABCA4, HLA-A, HLA-B, HLA-C, IRAK4, NFκB1, TGFβ1, TREX1), while five are down-modulated (ALOX12, COL2A1, P2RX7, TIRAPMAL, TLR9). In the globus pallidus that regulates voluntary movement, the susceptibility genes, other than ABCA4, ALOX12, COL2A1, NOD2, TIRAP, TLR9, are upregulated.

Upstream regulator analysis using IPA elucidates statistically significant upstream regulators including molecules which alter expression of downstream molecules. The systems analysis of upstream regulators of our susceptibility genes (Fig. 2b) underscores how this intracellular parasite influences the intricate balance between growth and death of its host’s cells. Upstream regulatory networks associated with these genes by IPA (Fig. 2b) include human genes that participate in inflammation, cell death, and cytokine signaling, as well as genetic, neurologic, and retinal diseases.

The transcriptomics analyses demonstrate some effects that are similar and some that differ between cell types (Figs 3 and 4). Possible explanations of the differences between cells include a number of variables including differences in the genetics of the host cells, fundamental differences in the cell types’ basal transcriptomics(Fig. S1), differences in culture conditions including media, growth factors and timing when cells were studied, or different responses to parasites. We also noted differences in responses when we studied another parasite called EGS which grows as encysted bradyzoites in tissue culture in HFF, MM6, and S-NSCs. Pathways that are perturbed suggest profound effects on host cells. These include for S-NSC: translational elongation, apoptosis, cell cycle, vesicle mediated transport, ribosomes, amino acid metabolism, TGF-β signaling, p53 signaling, MAP kinases, circadian rhythm, and cell cycle. For L-NSC: sensory perception of smell, mitochondrial organization, protein modification by small protein conjugation, cognition, neurologic system processes, neddylation, oxidative phosphorylation, G protein coupled receptor protein signaling, androgen and estrogen metabolism, ribosomes, translational elongation, and particularly noteworthy, pathways of Parkinson’s, Alzheimer’s and Huntington’s diseases. For MM6: modulation of P53 signaling, JAK-STAT signaling, programed cell death, arachidonic acid metabolism, response to hypoxia. Further, many of the perturbed miRNAs in the transcriptome have been associated with different cancer types, neurodegenerative diseases and the NFkB activation pathway. For instance, both infected S-NSC and S-NDC cells overexpressed mir-139, a microRNA that is overexpressed in the hippocampus of a mouse model for Alzheimer’s Disease (AD) and associated with impaired hippocampus-dependent learning and memory57. In addition, S-NSC cells infected with T. gondii PRU tachyzoites had more than a two-fold reduction in the expression of mir-29a and mir-107, found to be down-regulated in patients with AD58,59,60,61. mir-132, that is under-regulated in post-mortem Huntington’s disease patients and in a mouse model for this disease62, showed a ~four-fold down-regulation in GT1 and PRU-infected S-NSC cells. Expression of other miRNA molecules, that have been associated with the NFκB network and cancer, was also perturbed in S-NSC infected cells, such as mir-218, mir-143, mir-155, mir-199a, mir-21, miR-16 and mir-181b-163,64,65,66,67,68,69,70,71.

In the quantitative proteomics with L-NSC (Fig. 5a), ATXN2L, FXR1, and NPC2 were modulated. ATXN2L is a paralog of Ataxin 2, a protein that causes spinocerebellar ataxia type 272. It has been shown that ATXN2L is functionally similar to Ataxin 2 with respect to RNA metabolism and also plays a role in the regulation of stress granules and processing bodies in mammalian cells73. Overexpression of FXR1, a member of the Fragile X-related family of RNA-binding proteins, has been associated with suppression of cellular senescence and cancer74. NPC2 regulates the transport of cholesterol through the late endosomal/lysosomal system and mutations in this gene have been associated with Niemann-Pick disease type C2, a disease with a broad range of visceral, neurological and psychiatric clinical presentations75.

In the quantitative proteomics of S-NSC (Fig. 5b) many more genes transcripts were modulated. Some of particular interest are shown in Fig. 5b as follows: WDFY1 and PPP4C, two proteins known to modulate NFκB activity, a key factor to control parasite infections, were downregulated in infected S-NSC (Fig. 5b). WDFY1 induces TLR3- and TLR4-mediated activation of NFκB and the production of type I interferons and inflammatory cytokines76, while PPP4C is the catalytic subunit of protein phosphatase 4, a protein implicated in the activation of NFκB-mediated transcription77. This strongly suggests that T. gondii inhibits the human host NFkB pathway at multiple levels in addition to promoting p65 degradation through the virulence factor ROP 1878. Another two proteins, UBE3A and USP8, involved in protein ubiquitination, were also downregulated in infected S-NSC (Supplement B: Table S15). Interestingly, deubiquitin USP8 regulates the turnover of the epidermal growth factor receptor (EGFR) and its hyperactivation has been associated to constitutive EGFR-signaling leading to corticotroph tumorigenesis79. USP8 also regulates parkin-mediated mitophagy, a process believed to be central to the pathogenesis of Parkinson’s disease80, 81. Loss of function of the HECT-type E3 ubiquitin ligase UBE3A leads to Angelman syndrome, characterized by microcephaly, severe developmental delay, ataxia, seizures, and happy disposition82. Decrease expression of UBE3A has also been observed in Rett syndrome patients83. Within the proteins that were upregulated during S-NSC infection, was eEF1A2, a translation elongation factor that has been proposed to play a significant role in tumorigenesis and as an anti-apoptotic factor84,85,86 (Table S15). Pathways of cell death, TOR, protein transport/localization, RNA splicing, alternative splicing, oxidation reduction, vesicle mediated transport, iron homeostasis, glucose metabolism are noteworthy in this proteomics analysis.

We found it remarkable that we identified serum biomarkers in the ill children compared with those who were well (Fig. 6a–c). In considering these biomarkers in serum, three of those miRNAs, mir-17, mir-18b and mir-19a, are all encoded by the miR-17-92 family of miRNA clusters that modulate a number of protein-coding genes implicated in apoptosis, cell proliferation and angiogenesis87 (Fig. 6d). One of these miRs88, mir-17, is over-expressed in Human Foreskin Fibroblast cultures infected with the RH strain of T. gondii89. Also noteworthy, mir-124 associated with neurodegeneration90, was increased in sera of the three ill children (Fig. 6b,c). In addition, proteomics identified that ill children compared with their paired healthy controls had increases or decreases in certain serum proteins. Elevated proteins included clusterin (CLU)91,92,93, serum amyloid P-component (APCS)94, 95, and oxytocin(OXT)96,97,98,99 (Fig. 6d,e)91, 92, 95, 100,101,102,103,104,105,106. PGLYRP2 (Peptidoglycan recognition protein 2, with N-acetylmuramoyl-L-alanine amidase activity), that degrades an innate immunity recognition factor for peptidoglycans, was decreased in three of the ill children in the pairs (Fig. 6d). Apolipoprotein A1 also was decreased in the ill children (Fig. 6). These proteins are known to be associated with neurodegeneration. Specifically, clusterin is a chaperone which is increased in neurodegenerative diseases. It aids protein folding of secreted proteins, with three isoforms that are differently involved in pro- or anti-apoptotic processes. Thus this protein is involved in many diseases where there is oxidative stress including neurodegenerative diseases and aging. It is associated with Lewy bodies in Parkinson’s disease, with the pathology in Alzheimer’s disease and multiple system atrophy, Cerebrospinal fluid levels of clusterin may reflect pathology in neurodegenerative disease. Amyloid P is in amyloid fibrils and protects them from degradation, thus contributing to neurodegeneration in Alzheimer’s disease94. It is also an acute phase reactant. Oxytocin was present in the sera of the ill boys. Oxytocin diminishes inflammation, decreases anxiety, increases trust and empathy and mutations have been associated with autism spectrum disorder96,97,98,99. Hypothalamic cells produce oxytocin which is then secreted into the bloodstream by the posterior pituitary gland96,97,98,99. Secretion occurs when there is electrical activity, excitation, of hypothalamic neurons96,97,98,99. These findings suggest active brain destruction by the parasite or the response to it. These circulating miRNAs and proteins might prove clinically useful biomarkers to identify active toxoplasmic brain (or possibly retinal) disease if confirmed with more children’s sera correlated with their clinical findings.

Key upstream regulators were identified as shown in Figs. 7 and 8. Descriptions of target and upstream regulatory genes analyzed for L-NSC, cohort genetics and cohort biomarkers considered together are illustrated in Fig. 7. Key regulators we have found herein have been demonstrated to have significance in earlier work with other cell types: Those known empirically include HIF1α/VEGF107, 108 and others such as EGFR109 (Fig. 7; Details in Supplement B Table S7). IPA annotations of the total brain infectome and upstream regulatory bundle includes core (Supplement B: Table S7) and comparison (Supplement B: Table S7) analyses of target genes, upstream regulators and both. The convergence of the genes, biomarkers, trancriptomic data, proteomic data on the upstream regulators seen in this orbital diagram makes a model of the infectome which can be further empirically tested. T. gondii molecules which modify them then can be identified. Upstream regulatory genes such as JUN, MYC, EGFR, and VEGF110 provide examples of genes already known to be modulated by specific parasite proteins in other cell types109, 111,112,113. The biologic relevance of this finding for humans is evident from the observation that VEGF is very important in choroidal neovascular membranes that occur in clinical toxoplasmic chorioretinitis. These resolve when treated with antibody to VEGF administered in conjunction with anti-T. gondii medicines108.

Upstream regulatory genes contributing to the signature pathways with important biologic impact were compared when both L-NSC and S-NSC transcriptomic and proteomic data were analyzed together (Fig. 8). Noteworthy upsteam regulators were identified. For example, fibroblast growth factor114 and its receptors (e.g. FGRR1115), TGF-β, as well as the, ERK genes, PI3K, FoxOs116, and GM-CSF47, 117 are all involved in developmental and adult neurogenesis. Both TGF-b118 and PI3K/Akt119 are involved in ROS and inflammation-related actions during normal and pathological neurogenesis. In addition, TGF-β has been implicated in normal neural stem/progenitor cell growth and differentiation, as well as in cancer stem cell-mediated gliomagenesis120. Likewise, ERK signaling121 is involved in normal neural stem/progenitor cell fate choice during development. Another stem cell pathology involving PI3K/PTEN122 has shown that altering this pathway can result in interneuronal dysplasia and leukodystrophy as a result of altering neuronal and oligodendrocyte differentiation. All of these abnormal neurogenic phenotypes were present in the infected adult human neural progenitor cell population studied.

Cluster protein interactions of L-NSC total infectome provided profound insight into pathogenic mechanisms (Fig. 9). The functional clusters included cellular movement and migration important in mechanisms of immune and endocrine signaling, leucocyte migration, parasitism of lipid metabolism, ubiquitin-mediated protein degradation in cell cycle control, and hijacking protein synthesis in the cell cycle, all clustering around NEDD8123,124,125. As with ubiquitin and SUMO, NEDD8123,124,125 is conjugated to cellular proteins after the C-terminal tail is processed. T. gondii tachyzoites thereby alter host cell protein stability and degradation, potentially contributing to ER “stress” and the misfolded protein response associated with neurodegeneration. Other clusters include modulation of brain ATP production by mitochondrial oxidative phosphorylation. Another cluster shows effect on olfactory receptors (Fig. 9) suggesting a mechanism whereby parasites alter host sense of smell as seen in attraction of rodents and chimpanzees to cat urine56, 126. Parasitic modulation of these essential functions of the brains could lead to a wide range of diseases, including those discussed below (Fig. 10).

One NSC phenotype suggested by our genetics and omics analyses led us to study phenotypic effect of T. gondii isolates on NFκB in S-NSC with IFA (Fig. 11). Effect on NFκB noted in murine cell lines, macrophages and human fibroblasts and primary monocytes by others34, 36 were found mediated by Type II parasite dense granule protein (GRA) 15, whereas we found T. gondii infection of primary human neuronal stem cells by all three strains alters localization of NFκB (Fig. 11a). In other cell lines, STAT3 localization was modulated only by parasite Type I ROP16127,128,129. This is similar, but not identical, to the nuclear translocation of STAT3 in our human neuronal stem cells, which was increased most, but not exclusively, by the Type I strain. There was some similar effect for Type II and III strains (Fig. 11a). Effects on such cytokine signaling pathways have potential to contribute to maternal cytokine effects130 on fetal brain.

Another phenotype we selected for study included presence and localization of neurotransmitters (e.g., dopamine) and an enzyme in the brain involved in synthesis of this neurotransmitter, tyrosine hydroxylase. As shown in Fig. 11, we found T. gondii affects both dopamine and tyrosine hydroxylase and T. gondii tachyzoites contain dopamine. Disruption of neurotransmission is associated with epilepsy5, 131. Alteration of neurotransmitters is consistent with association studies of seropositivity and human epilepsy131,132,133,134, and three separate studies of mice4, 5, 131. These results are consistent with theories of T. gondii’s effects on reward pathways and depletion of serotonin by precursor tryptophan starvation9, 135, 136. These findings concerning neurotransmitters may support alterations of infectomes caused by the parasite affecting behavior, but these are predictions based on putative mechanisms observed in cells in tissue culture and remain to be confirmed empirically with experiments in vivo.

In other diseases, such as certain genetic diseases with repetitive DNA sequences, alternative splicing or mis-coding of transcripts can lead to truncated or misfolded proteins that are central causes of neurodegenerative diseases137. Perturbing genes associated with the misfolded protein response, and protein degradation seen in the IPA analyses, as well as inflammation are mechanisms whereby T. gondii may contribute to neurodegeneration, alterations of cell cycle/replication and death, and epilepsy4, 133, 134, 138 (Figs 10 and 11).

When we place our findings presented herein in the broad context of diseases and mechanisms of specific diseases, our work indicates T. gondii can cause a dominant alteration of the cell cycle and opposing regulation of cell growth and death. For example, as discussed above, in infected L-NSC, transcriptomes also showed that parasites modulate pathways of cell death, apoptosis and neddylation. Modulation in p53 signaling, ribosomes, amino acid metabolism, axon guidance, JAK-STAT, TGF-β, and cell cycle are especially noteworthy in the KEGG pathways for S-NSC (Fig. 2c). Transcriptomic analysis of L-NSC reveals pathways associated with Alzheimer’s, Parkinson’s and Huntington diseases and disruption of oxidative phosphorylation. Also alternative splicing pathways which might cause protein misfolding and neurologic diseases are affected (Fig. 5). Transcriptomics, proteomics, and cluster deconvolution identified alterations in neddylation and ubiquitination key in clearing misfolded proteins, neuronal cell viability, and synaptic plasticity (Figs 3–5, 9). We are investigating whether T. gondii causes alternative splicing which can be a central cause of protein misfolding, neurodegeneration, and other complex diseases137.

Although there are literature reports of associations between T. gondii seropositivity and schizophrenia in individual studies and in a meta-analyses of 38 studies139, there is no proven causality. An increase in dopamine metabolism is one possible pathologic mechanism140, 141. We detected dopamine and tyrosine hydroxylase immunostaining in the cytoplasm of T. gondii tachyzoites (Type I, II, and III) in S-NSC (Fig. 11). Alteration in dopamine neurotransmission has wide implications in behaviors and diseases, including epilepsy, neurodegeneration and movement disorders142. However, we do not identify any congenitally infected persons or their mothers with schizophrenia in our NCCCTS cohort (unpublished observations). Genetics and epigenetics of neuronal stem cells may be derived from donors who are not predisposed to schizophrenia. Primary cultures of NSC were temporal lobe tissues transected from patients who have epilepsy.

Cancer is the largest disease correlate to our T. gondii brain infectome. One explanation may be relative robustness of cancer research in comparison to other diseases in literature-based analyses. Some population studies show correlation with brain cancer. There are anecdotal descriptions of lymphomas developing in eyes of those with recurrent Toxoplasmic retinal disease143, 144. Targeted genetics studies of these T. gondii induced cancer genes and pathways might reveal higher penetrance. A strong argument for a T. gondii-cancer link is long known protection against tumor cells in murine models145. Recently, injection of attenuated, non-replicating parasites increased long-term survival of mice with melanoma146, pancreatic147, and ovarian148 cancers by stimulating high-level expression of co-stimulatory molecules CD80, CD86, IL-12, and tumor antigen specific CD8 + T cell populations and increasing cytolytic capacity of activated macrophages149. T. gondii may effect control of tumor growth and clearance through a network of 1,178 genes we have identified. Furthermore, our data may illuminate likely ways T. gondii may affect cancer stem cells, including stemness pathways of Wnt, TGF-β, STAT, among others150 and potential associations with Alzheimer’s disease. Some population serologic studies show conflicting correlations for151, 152 and against153, 154 T. gondii as a risk factor of Alzheimer’s disease or memory impairment. The parasite, however, inhibits neuronal degeneration as well as learning and memory impairments by immunosuppression in a murine model of Alzheimer’s disease155. T. gondii causes epilepsy131, possibly by altering GABAergic signaling6. Our analysis provides the systems map to study these correlations and applications of T. gondii as an immunotherapeutic tool.

Olfactory dysfunction is reported in Alzheimer’s disease and schizophrenia156. T. gondii increases cat predation of an infected rat by altering neural activity in limbic brain areas to block innate aversion of rats for cat urine. Infected chimpanzees lose their innate aversion towards urine of their natural predator, leopards126. There is a report that T. gondii might alter olfactory preferences in humans157. Our cluster deconvolution discovered 12 olfactory receptors in humans modulated by the brain parasite possibly causing olfactory preferences and dysfunction.

Only a subset of people with T. gondii infection, at the outset, have some of the diseases, like epilepsy or malignancy, that share the alterations in the signature pathways we identified. Our data indicate these signature, shared, molecular/cellular pathways, including inflammation, protein misfolding and mis-splicing, are altered by T. gondii. We found that effects differ in some cases for parasites with differing genetics and cells of different types or from different people. Our results provide insight into mechanisms whereby this parasite could cause these associated diseases under some circumstances. We show that tachyzoites, the rapidly growing form, can cause these158 alterations. In vivo this parasite interconverts from a dormant bradyzoite phase to an actively replicating tachyzoite phase and back again. This interconversion may be relevant to associations we recognized. Biomarkers we found were in ill children with new seizures and the two where we looked for this had reactivation of infection with activity documented at the time. In a separate parallel analysis of dormant parasites that form definitive cysts in vitro, similar pathways also were altered159 KEGG and GO analyses159 demonstrated a bradyzoite phenotype organism, called EGS, affects pathways involved in Alzheimer’s, Parkinson’s and Huntington’s diseases, splicing, and oxidative damage159. Thus, both tachyzoites and bradyzoites perturb critical host signaling pathways in common with those perturbed in epilepsy, neurodegenerative diseases, motor diseases including movement disorders, and brain cancer.

In neurodegenerative diseases and cancer, we have also shown glioma-initiating cancer stem-like cells possess an altered stem cell/developmentally regulated gene and protein mutanome160,161,162,163. An Adult Human Neural Progenitor Cell (AHNP)49, has been suggested to be susceptible to chronic microenvironmental inflammation in Parkinson’s Disease where it exhibits aberrant growth and differentiation164; in Alzheimer’s165, other neurodegenerative diseases and brain cancer. There are common growth-related programs and mutanomes in astrocytes and neurogenic astrocytes166 that are at-risk for transformation during chronic inflammation. Altered levels of cytokines and other immune- and inflammation-associated pathways, following T. gondii infection, have potential to support neurodegeneration and neoplastic transformation. It is notable that Zika virus infections also have a profound destructive effect on neuronal progenitor cells167 that share a normal or cancerous neuropoietic (i.e. persistent neurogenic) role during brain development and in the adult brain, including in the hippocampus168, 169. We have shown that pathology in this cell population is also involved in epilepsy, memory disorders, and autism in an interactome based on the literature. Carter et al. described similar overlap in neurologic susceptibility genes and those T. gondii modulates, based on a literature analysis. This created an interactome of T.gondii infection and genes implicated in a variety of neurologic and other diseases9. These diseases included multiple sclerosis, neurodegenerative diseases, epilepsy, and malignancies9. Overlap in Carter et al.’s analysis with those genes and pathways that T. gondii perturbs is striking9, although it is possible that they all involve the same pathways without T. gondii causing the diseases. A large data analysis of insured U.S. patients revealed the same associations with these diseases without clear directionality10.

All of these findings together support the notion that the dormant parasite, which sometimes interconverts to active tachyzoites when cysts rupture, and are present in chronic T. gondii infection in the brain of 2 billion persons, also has potential to contribute to these disease pathways. We identify some of the same mechanisms with tachyzoites. There is growing evidence for central nervous system transcellular spread of toxins, viruses, lectins and macromolecules including pathological proteins and nucleic acids. This serves to transmit such infectious elements170, 171 to naïve cells in neurodegenerative diseases (e.g., Parkinson’s and Alzheimer’s diseases) and cancer. In these diseases particular neural sites and associated circuitries are hijacked and also affect the same neural stem/progenitor cell studied here. We found in primary, T. gondii infected human neuronal stem cells these circuitries are altered in pathways of neurodegeneration, motor disease, movement disorders, epilepsy, malignancy and in odorant receptors. Neddylation, ubiquitination, alternative splicing, cell replication, and autophagy are altered, among others. These alterations provide some predicted mechanisms for the various clinical disease associations that also have been noted by others51, 138, 172,173,174,175,176,177,178,179,180,181.

Human chronic diseases are a complex interplay of genetic and environmental factors9, 136, 182,183,184 (Figs 1 and 10) requiring modified approaches to reconstruct their multifactorial etiology and cascades of developmental-plasticity mechanisms in precipitating disease. Human brain parasitism by T. gondii provides a model and template to examine development of brain diseases. This work provides a systems roadmap to design medicines and vaccines to repair and prevent neuropathologic effects of T. gondii infection of the human brain. Further, our original template provides a novel method to integrate multiple levels of intrinsic and extrinsic factors highlighting a way to unravel complexity in brain parasitism, toxoplasmosis specifically, and other complex diseases.