Sinusoidal obstruction syndrome associated with disseminated toxoplasmosis involving the liver after allogeneic hematopoietic stem cell transplantation: A case report.

Sinusoidal obstruction syndrome (SOS) is a fatal complication after hematopoietic stem cell transplantation (HSCT). Only a few complications after HSCT have been reported as risk factors for SOS, including sepsis. Here, we report the case of a 35-year-old male diagnosed with Philadelphia chromosome-positive acute lymphoblastic leukemia who underwent peripheral blood HSCT from a human leukocyte antigen-matched unrelated female donor in remission. Graft-versus-host disease prophylaxis contained tacrolimus, methotrexate, and low-dose anti-thymoglobulin. The patient was treated with methylprednisolone for engraftment syndrome from day 22. On day 53, he presented worsening fatigue, breathlessness, and abdominal pain in the right upper quadrant that had persisted for 4 days. Laboratory tests showed severe inflammation, liver dysfunction, and positive for Toxoplasma gondii PCR. He died on day 55. An autopsy showed SOS and disseminated toxoplasmosis. Hepatic infection with T. gondii was identified in zone 3 of the liver, which overlapped with the pathological features of SOS. In addition, the timing of the exacerbation of hepatic dysfunction coincided with the onset of systemic inflammatory symptoms and T. gondii reactivation. This rare case of toxoplasmosis is the first to suggest that hepatic infection with T. gondii is strongly associated with SOS after HSCT.

New PCR primers targeting the cytochrome b gene reveal diversity of Leucocytozoon lineages in an individual host.

Avian haemosporidian parasites have received considerable attention in ecology and evolution as a result of their wide distribution and ease of detection. However, conventional PCR-based detection methods may sometimes underestimate haemosporidian mixed infections, which are frequent in natural populations. This underestimation is due to differences in PCR sensitivity for detection of lineages within the mixed infections. Therefore, we designed new primers to amplify sequences that were not detected by the conventional primers and examined if our primers were useful for accurate detection of mixed infections. Blood samples were collected from 32 wild birds captured in Hokkaido, and 16 of these were positive for Leucocytozoon using the conventional primers, while 15 were positive using our primers. All positively amplified samples were sequenced, and we found that the conventional primers detected 16% (5/32) of multiple infections and none of them was a novel lineage, whereas our primers detected 44% (14/32) of multiple infections and ten of them were novel lineages. A phylogenetic analysis showed that the new primers can detect a wide range of Leucocytozoon lineages compared with that detected by the conventional primers. The results indicate that our primers are particularly suitable for revealing unique strains from multiple infections. Highly variable multiple infections in the same population of birds at the same location were found for the first time. We revealed a higher diversity of Leucocytozoon lineages in nature than expected, which would provide more information to better understand parasite diversity and host-vector interactions in wildlife.

Clinical characteristics and incidence of toxoplasmosis after autologous hematopoietic stem cell transplantation: A retrospective study and literature review.

BACKGROUND: Toxoplasmosis is a rare but life-threatening infection occurring in immunocompromised hosts, including allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. However, thus far, the clinical features and incidence of toxoplasmosis in autologous HSCT (auto-HSCT) recipients remain unknown. This retrospective survey aimed to analyze 152 patients who received auto-HSCT between 1998 and 2017. METHODS: Serological tests for Toxoplasma gondii-specific IgG were performed on 109 (71.7%) recipients, and 12 pre-HSCT recipients (11%) were Toxoplasma seropositive. Among the 12 recipients, three who did not receive trimethoprim-sulfamethoxazole (TMP/SMX) prophylaxis developed cerebral, pulmonary or disseminated toxoplasmosis due to reactivation after auto-HSCT and died despite treatment. RESULTS: The incidences of toxoplasmosis were 2% and 25% among 152 auto-HSCT recipients (five recipients received auto-HSCT two times) and 12 pre-HSCT Toxoplasma seropositive recipients, respectively. Further, we conducted a literature review and identified 21 cases of toxoplasmosis following auto-HSCT. In these previous cases, the mortality rate was high, especially for pulmonary and disseminated toxoplasmosis. Our findings suggest that, similar to toxoplasmosis after allo-HSCT, toxoplasmosis after auto-HSCT is a fatal complication. CONCLUSIONS: Serial screening of T. gondii-specific IgG before HSCT could contribute to the detection of Toxoplasma reactivation and allow for prompt diagnosis and treatment. The present study is the first to reveal the incidence of toxoplasmosis after auto-HSCT among seropositive patients in Japan.

Genetic polymorphisms in malaria vaccine candidate Plasmodium falciparum reticulocyte-binding protein homologue-5 among populations in Lagos, Nigeria.

BACKGROUND: Vaccines are the most reliable alternative to elicit sterile immunity against malaria but their development has been hindered by polymorphisms and strain-specificity in previously studied antigens. New vaccine candidates are therefore urgently needed. Highly conserved Plasmodium falciparum reticulocyte-binding protein homologue-5 (PfRH5) has been identified as a potential candidate for anti-disease vaccine development. PfRH5 is essential for erythrocyte invasion by merozoites and crucial for parasite survival. However, there is paucity of data on the extent of genetic variations on PfRH5 in field isolates of Plasmodium falciparum. This study described genetic polymorphisms at the high affinity binding polypeptides (HABPs) 36718, 36727, 36728 of PfRH5 in Nigerian isolates of P. falciparum. This study tested the hypothesis that only specific conserved B and T cell epitopes on PfRH5 HABPs are crucial for vaccine development. METHODS: One hundred and ninety-five microscopically confirmed P. falciparum samples collected in a prospective cross-sectional study of three different populations in Lagos, Nigeria. Genetic diversity and haplotype construct of Pfrh5 gene were determined using bi-directional sequencing approach. Tajima's D and the ratio of nonsynonymous vs synonymous mutations were utilized to estimate the extent of balancing and directional selection in the pfrh5 gene. RESULTS: Sequence analysis revealed three haplotypes of PfRH5 with negative Tajima's D and dN/dS value of - 1.717 and 0.011 ± 0.020, respectively. A single nucleotide polymorphism, SNP (G → A) at position 608 was observed, which resulted in a change of the amino acid cysteine at position 203 to tyrosine. Haplotype and nucleotide diversities were 0.318 ± 0.016 and 0.0046 ± 0.0001 while inter-population genetic differentiation ranged from 0.007 to 0.037. Five polypeptide variants were identified, the most frequent being KTKYH with a frequency of 51.3%. One B-cell epitope, 151 major histocompatibility complex (MHC) class II T-cell epitopes, four intrinsically unstructured regions (IURs) and six MHC class I T-cell epitopes were observed in the study. Phylogenetic analysis of the sequences showed clustering and evidence of evolutionary relationship with 3D7, PAS-2 and FCB-2 RH5 sequences. CONCLUSIONS: This study has revealed low level of genetic polymorphisms in PfRH5 antigen with B- and T-cell epitopes in intrinsically unstructured regions along the PfRH5 gene in Lagos, Nigeria. A broader investigation is however required in other parts of the country to support the possible inclusion of PfRH5 in a cross-protective multi-component vaccine.

Desensitization to trimethoprim-sulfamethoxazole in a toxoplasmic encephalitis patient who was intolerant to conventional treatments.

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmic encephalitis (TE) in immunocompromised patients. We describe a case of a 29-year-old Japanese man presenting with headache and vomiting. He had previously been diagnosed with human immunodeficiency virus infection. Magnetic resonance imaging identified some nodules in his brain. We suspected TE and began treatment successively with parenteral trimethoprim-sulfamethoxazole (TMP/SMX) plus clindamycin. After that, we switched to pyrimethamine plus sulfadiazine (PMT/SDZ) because these drugs are the first-line treatment for TE. Because the patient experienced nausea and vomiting, PMT/SDZ was replaced with TMP/SMX, atovaquone, and clindamycin. However, the patient could not tolerate them owing to their adverse reactions. Thus, we attempted oral desensitization to TMP/SMX to treat his TE. We began desensitization with 0.4/2 mg of TMP/SMX. The patient experienced morbilliform rash and elevated aminotransferase levels. Therefore, we administered a glycyrrhizin and an antihistamine and continued the last tolerable dose until these symptoms improved. After 37 days, we achieved desensitization to 160/800 mg of TMP/SMX, and the patient's symptoms improved. After using nested-polymerase chain reaction to identify T. gondii DNA in his frozen cerebrospinal fluid, which was collected at admission, his diagnosis was confirmed as TE. This might be the first case to attempt desensitization to TMP/SMX to treat TE.

The complete mitochondrial genome of Sarcoptes scabiei var. nyctereutis from the Japanese raccoon dog: Prediction and detection of two transfer RNAs (tRNA-A and tRNA-Y).

Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.

[Toxoplasmosis-associated central nervous system vasculitis accompanied by multiple cerebral hemorrhages developing subsequent to cord blood transplantation].

A 57-year-old man with high-risk myelodysplastic syndrome underwent umbilical cord blood transplantation. He began receiving steroids on day 14 for acute graft-versus-host disease, and experienced dizziness on day 75 during gradual dose reduction. Multiple hemorrhages were observed in the cerebrum, cerebellum, and brainstem. His bleeding increased, and he underwent a brain biopsy on day 91. Subsequently, he was diagnosed with central nervous system vasculitis (CNSV) on the basis of the observed aggregation of mature CD3+ lymphocytes around small vessels and vascular wall invasion by lymphocytes and macrophages. After receiving high-dose steroid therapy, cerebral hemorrhage stopped; however, dysphasia occurred on day 113 and the patient died of cerebral edema on day 128. Toxoplasma DNA and tachyzoites were detected in the brain biopsy specimen during additional examinations; therefore, we suspected that the toxoplasmosis was related to the onset of CNSV. CNSV is a rare, rapidly progressing disease that may present as a fatal post-transplantation central nervous system complication. Investigating the causes of CNSV, including CNSV associated with toxoplasmosis, is critically important for improving the prognosis of patients with CNSV.

Early diagnosis and successful treatment of disseminated toxoplasmosis after cord blood transplantation.

A 66-year-old woman with refractory angioimmunoblastic T-cell lymphoma underwent cord blood transplantation. Prior to transplantation, a serological test for Toxoplasma gondii-specific IgG antibodies was positive. On day 96, she exhibited fever and dry cough. Chest CT showed diffuse centrilobular ground glass opacities in both lungs. The reactivation of T. gondii was identified by the presence of parasite DNA in peripheral blood and bronchoalveolar lavage fluid. Moreover, brain MRI revealed a space occupying lesion in the right occipital lobe. Therefore, disseminated toxoplasmosis was diagnosed. She received pyrimethamine and sulfadiazine from day 99. The lung and brain lesions both showed improvement but the PCR assay for T. gondii DNA in peripheral blood was positive on day 133. On day 146, she developed blurred vision and reduced visual acuity, and a tentative diagnosis of toxoplasmic retinochoroiditis was made based on ophthalmic examination results. As agranulocytosis developed on day 158, we decided to discontinue pyrimethamine and sulfadiazine and the treatment was thus switched to atovaquone. Moreover, we added spiramycin to atovaquone therapy from day 174, and her ocular condition gradually improved. In general, the prognosis of disseminated toxoplasmosis after hematopoietic stem cell transplantation (HSCT) is extremely poor. However, early diagnosis and treatment may contribute to improvement of the fundamentally dismal prognosis of disseminated toxoplasmosis after HSCT.

In vivo curative and protective potential of orally administered 5-aminolevulinic acid plus ferrous ion against malaria.

5-Aminolevulinic acid (ALA) is a naturally occurring amino acid present in diverse organisms and a precursor of heme biosynthesis. ALA is commercially available as a component of cosmetics, dietary supplements, and pharmaceuticals for cancer diagnosis and therapy. Recent reports demonstrated that the combination of ALA and ferrous ion (Fe(2+)) inhibits the in vitro growth of the human malaria parasite Plasmodium falciparum. To further explore the potential application of ALA and ferrous ion as a combined antimalarial drug for treatment of human malaria, we conducted an in vivo efficacy evaluation. Female C57BL/6J mice were infected with the lethal strain of rodent malaria parasite Plasmodium yoelii 17XL and orally administered ALA plus sodium ferrous citrate (ALA/SFC) as a once-daily treatment. Parasitemia was monitored in the infected mice, and elimination of the parasites was confirmed using diagnostic PCR. Treatment of P. yoelii 17XL-infected mice with ALA/SFC provided curative efficacy in 60% of the mice treated with ALA/SFC at 600/300 mg/kg of body weight; no mice survived when treated with vehicle alone. Interestingly, the cured mice were protected from homologous rechallenge, even when reinfection was attempted more than 230 days after the initial recovery, indicating long-lasting resistance to reinfection with the same parasite. Moreover, parasite-specific antibodies against reported vaccine candidate antigens were found and persisted in the sera of the cured mice. These findings provide clear evidence that ALA/SFC is effective in an experimental animal model of malaria and may facilitate the development of a new class of antimalarial drug.

Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Genetic relatedness of third-generation cephalosporin-resistant Escherichia coli among livestock, farmers, and patients in Japan.

Objectives: The third-generation cephalosporin (3GC)-resistant E. coli strains have been detected worldwide in humans and animals. Hence, in this study, we evaluated the prevalence and genetic characteristics of 3GC-resistant E. coli in livestock, farmers, and patients to further analyse if livestock serves as a potential reservoir of antimicrobial-resistant bacteria. Methods: Faecal samples were collected from 330 healthy livestock (216 cattle and 114 swine), 61 healthy livestock farmers (52 cattle farmers and 9 swine farmers), and 68 non-duplicate 3GC-resistant E. coli isolates were also obtained from the clinical specimens of patients in Japan between 2013 and 2015. Genes associated with resistance in 3GC-resistant E. coli were identified using polymerase chain reaction (PCR) and DNA sequencing. Genotypic diversity was determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results: We obtained 39 and 17 non-duplicated 3GC-resistant E. coli strains from healthy livestock (33 cattle and six swine) and livestock farmers, respectively. All isolates carried either CTX-M-type extended-spectrum ß-lactamase or plasmid-mediated AmpC ß-lactamase genes, with CTX-M-14 being the most frequent. CTX-M producers from livestock and patients belonged to 22 and 19 different sequence types (STs), respectively, and only three STs were the same. Among the 3GC-resistant E. coli from livestock and farmers, three types of CTX-M producers have shown similar characteristics (CTX-M genotype, ST, PFGE patterns, and antimicrobial susceptibilities) and were identified as clonal isolates shared among their farms. Conclusions: Our study findings indicate that CTX-M-14 is predominant in Japan. No distinct relationship was observed between the 3GC-resistant E. coli isolated from livestock and patients; however, some clonal relatedness was observed between the isolates from livestock and farmers due to their close contact.

Novel type of linear mitochondrial genomes with dual flip-flop inversion system in apicomplexan parasites, Babesia microti and Babesia rodhaini.

BACKGROUND: Mitochondrial (mt) genomes vary considerably in size, structure and gene content. The mt genomes of the phylum Apicomplexa, which includes important human pathogens such as the malaria parasite Plasmodium, also show marked diversity of structure. Plasmodium has a concatenated linear mt genome of the smallest size (6-kb); Babesia and Theileria have a linear monomeric mt genome (6.5-kb to 8.2-kb) with terminal inverted repeats; Eimeria, which is distantly related to Plasmodium and Babesia/Theileria, possesses a mt genome (6.2-kb) with a concatemeric form similar to that of Plasmodium; Cryptosporidium, the earliest branching lineage within the phylum Apicomplexa, has no mt genome. We are interested in the evolutionary origin of linear mt genomes of Babesia/Theileria, and have investigated mt genome structures in members of archaeopiroplasmid, a lineage branched off earlier from Babesia/Theileria. RESULTS: The complete mt genomes of archaeopiroplasmid parasites, Babesia microti and Babesia rodhaini, were sequenced. The mt genomes of B. microti (11.1-kb) and B. rodhaini (6.9-kb) possess two pairs of unique inverted repeats, IR-A and IR-B. Flip-flop inversions between two IR-As and between two IR-Bs appear to generate four distinct genome structures that are present at an equi-molar ratio. An individual parasite contained multiple mt genome structures, with 20 copies and 2 - 3 copies per haploid nuclear genome in B. microti and B. rodhaini, respectively. CONCLUSION: We found a novel linear monomeric mt genome structure of B. microti and B. rhodhaini equipped with dual flip-flop inversion system, by which four distinct genome structures are readily generated. To our knowledge, this study is the first to report the presence of two pairs of distinct IR sequences within a monomeric linear mt genome. The present finding provides insight into further understanding of evolution of mt genome structure.

Siccanin Is a Dual-Target Inhibitor of Plasmodium falciparum Mitochondrial Complex II and Complex III.

Plasmodium falciparum contains several mitochondrial electron transport chain (ETC) dehydrogenases shuttling electrons from the respective substrates to the ubiquinone pool, from which electrons are consecutively transferred to complex III, complex IV, and finally to the molecular oxygen. The antimalarial drug atovaquone inhibits complex III and validates this parasite's ETC as an attractive target for chemotherapy. Among the ETC dehydrogenases from P. falciparum, dihydroorotate dehydrogenase, an essential enzyme used in de novo pyrimidine biosynthesis, and complex III are the two enzymes that have been characterized and validated as drug targets in the blood-stage parasite, while complex II has been shown to be essential for parasite survival in the mosquito stage; therefore, these enzymes and complex II are considered candidate drug targets for blocking parasite transmission. In this study, we identified siccanin as the first (to our knowledge) nanomolar inhibitor of the P. falciparum complex II. Moreover, we demonstrated that siccanin also inhibits complex III in the low-micromolar range. Siccanin did not inhibit the corresponding complexes from mammalian mitochondria even at high concentrations. Siccanin inhibited the growth of P. falciparum with IC50 of 8.4 µM. However, the growth inhibition of the P. falciparum blood stage did not correlate with ETC inhibition, as demonstrated by lack of resistance to siccanin in the yDHODH-3D7 (EC50 = 10.26 µM) and Dd2-ELQ300 strains (EC50 = 18.70 µM), suggesting a third mechanism of action that is unrelated to mitochondrial ETC inhibition. Hence, siccanin has at least a dual mechanism of action, being the first potent and selective inhibitor of P. falciparum complexes II and III over mammalian enzymes and so is a potential candidate for the development of a new class of antimalarial drugs.

Development of a novel electroporation method for the oyster parasite Perkinsus marinus.

Gene manipulation techniques are fundamental to molecular biology and are continuously being improved. However, gene transfection methods are not established for many unicellular eukaryotes (protists), thereby hindering molecular biological investigations. The oyster parasite Perkinisus marinus is one of the few protists with established gene transfection and drug selection. Nevertheless, the present protocols are tedious, requiring a specific electroporator and pulse conditions which limits the accessibility of this technique across different research groups. Here, we present alternative buffer and electroporation conditions that make the protocol less restrictive. We revealed the pulse condition that enables the introduction of plasmids into P. marinus cell using Ingenio electroporation buffer and NEPA21 electroporator. We found that number of cells and plasmid concentration were critical parameters for the electroporation system. We also constructed a simpler expression plasmid that is removed needless regions for gene expression in the parasite. Our findings resolved the equipment restriction in electroporation of P. marinus and would be a good reference for electroporation in other protists, in particular other Perkinsozoa parasites and core dinoflagellates.

Erdheim-Chester Disease Involving the Central Nervous System with Latent Toxoplasmosis.

Erdheim-Chester disease (ECD) is a rare, non-Langerhans cell histiocytosis characterized by the infiltration of foamy histiocytes into multiple organs. We herein report a case of ECD with central nervous system (CNS) involvement in a 63-year-old man who also presented a positive result for Toxoplasma gondii nested polymerase chain reaction testing of cerebrospinal fluid. Since anti-Toxoplasma treatment proved completely ineffective, we presumed latent infection of the CNS with T. gondii. This case suggests the difficulty of distinguishing ECD with CNS involvement from toxoplasmic encephalitis and the possibility of a relationship between the pathogeneses of ECD and infection with T. gondii.

Divergence of the mitochondrial genome structure in the apicomplexan parasites, Babesia and Theileria.

Mitochondrial (mt) genomes from diverse phylogenetic groups vary considerably in size, structure, and organization. The genus Plasmodium, causative agent of malaria, of the phylum Apicomplexa, has the smallest mt genome in the form of a circular and/or tandemly repeated linear element of 6 kb, encoding only three protein genes (cox1, cox3, and cob). The closely related genera Babesia and Theileria also have small mt genomes (6.6 kb) that are monomeric linear with an organization distinct from Plasmodium. To elucidate the structural divergence and evolution of mt genomes between Babesia/Theileria and Plasmodium, we determined five new sequences from Babesia bigemina, B. caballi, B. gibsoni, Theileria orientalis, and T. equi. Together with previously reported sequences of B. bovis, T. annulata, and T. parva, all eight Babesia and Theileria mt genomes are linear molecules with terminal inverted repeats (TIRs) on both ends containing three protein-coding genes (cox1, cox3, and cob) and six large subunit (LSU) ribosomal RNA (rRNA) gene fragments. The organization and transcriptional direction of protein-coding genes and the rRNA gene fragments were completely conserved in the four Babesia species. In contrast, notable variation occurred in the four Theileria species. Although the genome structures of T. annulata and T. parva were nearly identical to those of Babesia, an inversion in the 3-kb central region was found in T. orientalis. Moreover, the T. equi mt genome is the largest (8.2 kb) and most divergent with unusually long TIR sequences, in which cox3 and two LSU rRNA gene fragments are located. The T. equi mt genome showed little synteny to the other species. These results suggest that the Theileria mt genome is highly diverse with lineage-specific evolution in two Theileria species: genome inversion in T. orientalis and gene-embedded long TIR in T. equi.

Murine Model of Primary Acquired Ocular Toxoplasmosis: Fluorescein Angiography and Multiplex Immune Mediator Profiles in the Aqueous Humor.

Purpose: To establish a murine model of primary acquired ocular toxoplasmosis (OT) and to investigate the immune mediator profiles in the aqueous humor (AH). Methods: C57BL/6 mice were perorally infected with Toxoplasma gondii. The ocular fundus was observed, and fluorescein angiography (FA) was performed. The AH, cerebrospinal fluid (CSF), and serum were collected before infection and at 28 days post-infection (dpi); the immune mediator levels in these samples were analyzed using multiplex bead assay. Results: Fundus imaging revealed soft retinochoroidal lesions at 14 dpi; many of these lesions became harder by 28 dpi. FA abnormalities, such as leakage from retinal vessels and dilation and tortuosity of the retinal veins, were observed at 14 dpi. Nearly all these abnormalities resolved spontaneously at 28 dpi. In the AH, interferon-γ, interleukin (IL)-1α, IL-1ß, IL-6, IL-10, IL-12(p40), IL-12(p70), CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß, CCL5/RANTES, and CXCL1/KC levels increased after infection. All these molecules except IL-1α, IL-4, and IL-13 showed almost the same postinfection patterns in the CSF as they did in the AH. The tumor necrosis factor α, IL-4, and IL-5 levels in the AH and CSF of the T. gondii-infected mice were lower than those in the serum. The postinfection IL-1α, IL-6, CCL2/MCP-1, CCL4/MIP-1ß, and granulocyte colony-stimulating factor levels in the AH were significantly higher than those in the CSF and serum. Conclusions: A murine model of primary acquired OT induced via the natural infection route was established. This OT model allows detailed ophthalmologic, histopathologic, and immunologic evaluations of human OT. Investigation of AH immune modulators provides new insight into OT immunopathogenesis.

Acute exacerbation of pulmonary toxoplasmosis during corticosteroid therapy for immune thrombocytopenia: A case report and literature review.

RATIONALE: Pulmonary toxoplasmosis (PT) is an infectious disease that can be fatal if reactivation occurs in the recipients of hematopoietic stem cell transplantation (HSCT) who were previously infected with Toxoplasma gondii. However, whether the toxoplasmosis reactivation is an actual risk factor for patients receiving immunosuppressive therapies without HSCT remains unclear. Therefore, reactivated PT is not typically considered as a differential diagnosis for pneumonia other than in patients with HSCT or human immunodeficiency virus (HIV). PATIENT CONCERNS: A 77-year-old man presented with fever and nonproductive cough for several days. He was hospitalized due to atypical pneumonia that worsened immediately despite antibiotic therapy. Before 4 months, he was diagnosed with immune thrombocytopenia (ITP) and received corticosteroid therapy. Trimethoprim-sulfamethoxazole (ST) was administered to prevent pneumocystis pneumonia resulting from corticosteroid therapy. DIAGNOSIS: The serological and culture test results were negative for all pathogens except T. gondii immunoglobulin G antibody. Polymerase chain reaction, which can detect T. gondii from frozen bronchoalveolar lavage fluid, showed positive results. Therefore, he was diagnosed with PT. INTERVENTION: ST, clindamycin, and azithromycin were administered. Pyrimethamine and sulfadiazine could not be administered because his general condition significantly worsened at the time of polymerase chain reaction (PCR) examination. OUTCOMES: The patient died of acute respiratory distress syndrome despite anti-T. gondii treatment. An autopsy revealed a severe organizing pneumonia and a small area of bronchopneumonia. LESSONS: PT should be considered as a differential diagnosis in patients with pneumonia, particularly in seropositive patients who receive immunosuppressive therapies even for other than HSCT or HIV.

Hypericum perforatum extract and hyperforin inhibit the growth of neurotropic parasite Toxoplasma gondii and infection-induced inflammatory responses of glial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. has been widely used as a natural antidepressant. However, it is unknown whether it is effective in treating infection-induced neuropsychiatric disorders. AIM OF THE STUDY: In order to evaluate the effectiveness of H. perforatum against infection with neurotropic parasite Toxoplasma gondii, which has been linked to neuropsychiatric disorders, this study investigated the anti-Toxoplasma activity using in vitro models. MATERIALS AND METHODS: Dried alcoholic extracts were prepared from three Hypericum species: H. perforatum, H. erectum, and H. ascyron. H. perforatum extract was further separated by solvent-partitioning. Hyperforin and hypericin levels in the extracts and fractions were analyzed by high resolution LC-MS. Anti-Toxoplasma activities were tested in vitro, using cell lines (Vero and Raw264), murine primary mixed glia, and primary neuron-glia. Toxoplasma proliferation and stage conversion were analyzed by qPCR. Infection-induced damages to the host cells were analyzed by Sulforhodamine B cytotoxicity assay (Vero) and immunofluorescent microscopy (neurons). Infection-induced inflammatory responses in glial cells were analysed by qPCR and immunofluorescent microscopy. RESULTS: Hyperforin was identified only in H. perforatum among the three tested species, whereas hypericin was present in H. perforatum and H. erectum. H. perforatum extract and hyperforin-enriched fraction, as well as hyperforin, exhibited significant anti-Toxoplasma property as well as inhibitory activity against infection-induced inflammatory responses in glial cells. In addition, H. perforatum-derived hyperforin-enriched fraction restored neuro-supportive environment in mixed neuron-glia culture. CONCLUSIONS: H. perforatum and its major constituent hyperforin are promising anti-Toxoplasma agents that could potentially protect neurons and glial cells against infection-induced damages. Further study is warranted to establish in vivo efficacy.

Development of a rapid scabies immunodiagnostic assay based on transcriptomic analysis of Sarcoptes scabiei var. nyctereutis.

Scabies is a highly contagious skin disease caused by the mite Sarcoptes scabiei that affects many mammals. However, the sensitivity of traditional tests for scabies diagnosis in humans is less than 50%. To simplify the diagnosis of scabies, methods that are simple, sensitive, specific, and cost-effective are required. We developed an immunodiagnostic test based on S. scabiei var. nyctereutis RNA-seq data collected from Japanese raccoon dogs with sarcoptic mange. Three candidate antigens-a highly expressed hypothetical protein "QR98_0091190," another mite allergen known as "SMIPP-Cc," and an abundant "vitellogenin-like protein"-were evaluated by western-blot analysis. A lateral flow immunoassay, using specific antibodies against the vitellogenin-like protein, successfully detected scabies in the skin flakes of S. scabiei-infected raccoon dogs. This assay can potentially diagnose scabies more accurately in wildlife, as well as in humans.