A delayed diagnosis of fascioliasis: The importance of appropriate fecal diagnostic method.

Fascioliasis, a zoonotic helminthiasis, occurs sporadically in Japan. In this report, we describe a case of fascioliasis that was initially difficult to diagnose because the fecal examination method was negative for the Fasciola sp. eggs. A 64-year-old man living in Shimonoseki City, Japan, presented with fatigue and anorexia. Laboratory tests showed hepatic dysfunction and eosinophilia. Abdominal dynamic contrast-enhanced computed tomography and magnetic resonance cholangiopancreatography suggested intrahepatic biliary cysts. Thereafter, fever and night sweats persisted, and positron emission tomography and biopsy of the porta hepatis lymph node were performed on suspicion of malignancy. However, histopathological diagnosis found non-specific inflammation. As fascioliasis was suspected due to eosinophilia and the multiple hepatic masses, fecal egg examination was performed by an external private laboratory, which adopted the flotation method and reported the absence of parasite eggs. However, fecal examination was retried in our laboratory using the formalin-ether concentration method, and we detected Fasciola sp. eggs. This case suggests that misdiagnosis may occur depending on the fecal examination method; thus, it is necessary to choose a suitable method for certain parasite species.

Four successive cases of human fasciolosis in Japan.

Fasciolosis is a food-borne parasitic disease, caused by the large liver fluke, Fasciola. Humans acquire infection by ingesting fresh or undercooked water plants, on which infective metacercaria encyst. In spite of the rarity of the disease in Japan, we encountered four successive fasciolosis patients within a short period, who were all living in the same area. The patients were 70-82 years old, three females and the husband of one of the female patients. They started complaining of non-specific symptoms, such as fever, general fatigue, appetite loss, and abdominal pain, almost at the same time. All patients showed prominent peripheral blood eosinophilia, and the medical imaging indicated multiple hepatic lesions. No parasite eggs or worms were detected in any of the patients. Diagnosis was made serologically and they were treated with praziquantel and/or triclabendazole. No cattle or sheep were farmed in the area, but the wild sika deer, Cervus nippon, inhabited adjacent to the residential area. The intermediate host snail, Austropeplea ollula, were found near the residence of the patients, and one of the collected snails was positive for F. hepatica/gigantica hybrid type rediae. Our report should alarm the medical professionals for this rare and unfamiliar parasitic disease.

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration.

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.

A Cluster of Paragonimiasis with Delayed Diagnosis Due to Difficulty Distinguishing Symptoms from Post-COVID-19 Respiratory Symptoms: A Report of Five Cases.

Paragonimiasis caused by trematodes belonging to the genus Paragonimus is often accompanied by chronic respiratory symptoms such as cough, the accumulation of sputum, hemoptysis, and chest pain. Prolonged symptoms, including respiratory symptoms, after coronavirus disease 2019 infection (COVID-19) are collectively called post-COVID-19 conditions. Paragonimiasis and COVID-19 may cause similar respiratory symptoms. We encountered five cases of paragonimiasis in patients in Japan for whom diagnoses were delayed due to the initial characterization of the respiratory symptoms as a post-COVID-19 condition. The patients had consumed homemade drunken freshwater crabs together. One to three weeks after consuming the crabs, four of the five patients were diagnosed with probable COVID-19. The major symptoms reported included cough, dyspnea, and chest pain. The major imaging findings were pleural effusion, pneumothorax, and nodular lesions of the lung. All the patients were diagnosed with paragonimiasis based on a serum antibody test and peripheral blood eosinophilia (560-15,610 cells/µL) and were treated successfully with 75 mg/kg/day praziquantel for 3 days. Before diagnosing a post-COVID-19 condition, it is necessary to consider whether other diseases, including paragonimiasis, may explain the symptoms. Further, chest radiographic or blood tests should be performed in patients with persistent respiratory symptoms after being infected with COVID-19 to avoid overlooking the possibility of infection.

Primaquine plus clindamycin as a promising salvage therapy for Pneumocystis jirovecii pneumonia: A retrospective analysis in Japanese patients.

Treatment of intractable Pneumocystis jirovecii pneumonia (PCP) patients with primaquine (PQ) in combination with clindamycin (CLDM) was conducted by the Research Group on Chemotherapy of Tropical Diseases (RG-CTD), as a kind of compassionate use. Primaquine was not nationally licensed at the time but imported by RG-CTD for the use in a clinical research to investigate safety and efficacy in malaria treatment. Eighteen Japanese adult patients thus treated were analyzed. Prior to the treatment with PQ-CLDM, most of the patients had been treated with trimethoprim-sulfamethoxazole first, all of which being followed by pentamidine and/or atovaquone treatment. This combination regimen of PQ-CLDM was effective in 16 (89%) patients and developed adverse events (AEs) in five (28%) patients. AEs included skin lesions, methemoglobinemia, and hepatic dysfunction, though none of them were serious. As a second-line or salvage treatment for PCP, PQ-CLDM appears to be a better option than pentamidine or atovaquone. Currently in Japan, both PQ and CLDM are licensed drugs but neither of them is approved for treatment of PCP. Considering the potentially fatal nature of PCP, approval of PQ-CLDM for treating this illness should be urged.

Seroprevalence of toxoplasmosis among reproductive-aged women in Myanmar and evaluation of luciferase immunoprecipitation system assay.

BACKGROUNDS: Primary infection with Toxoplasma gondii during pregnancy can pose serious health problems for the fetus. However, the epidemiological status of toxoplasmosis among reproductive-aged population in Myanmar is largely unknown. Although luciferase immunoprecipitation system (LIPS) assays for serodiagnosis of toxoplasmosis was developed mostly using mouse infection model, had not been tested by using field-derived human samples. METHODS: A total of 251 serum samples were collected from reproductive-aged women, residing in Shwegyin township, Bago region, Myanmar and analyzed with a commercial ELISA kit, as well as in-house LIPS assays. RESULTS: The overall seroprevalence for Toxoplasma gondii infection by the commercial ELISA was 11.5%. No clear risk factor was identified except for being in the younger age group (15-30 years old). Overall, LIPS assays showed low sensitivity when the commercial ELSA was used as a reference test. CONCLUSION: We identified the epidemiological situation of toxoplasmosis in some rural communities in Myanmar. The data obtained here will serve as a primary information for the effort to reduce toxoplasmosis in this region. Although looked promising in the previous experiments with mouse infection model, we found that the reported LIPS procedures need further improvements to increase the sensitivities.

Severe delayed haemolytic anaemia associated with artemether-lumefantrine treatment of malaria in a Japanese traveller.

Delayed haemolytic anaemia has been reported in association with intravenous artesunate treatment in patients with severe Plasmodium falciparum malaria, and furthermore, oral artemisinin-based combination therapies including artemether-lumefantrine (AL) have also been incriminated. However, definite cases of delayed haemolytic anaemia associated with AL appear to be scarce, as reported cases were often treated concomitantly with other anti-malarials. In this study, we report a severe case of delayed haemolytic anaemia following AL alone in a Japanese traveller with severe parasitaemia caused by numerous P. falciparum parasites and a few P. vivax parasites. We also stress the need by further studies to differentiate between delayed haemolytic anaemia and blackwater fever, the latter being another malaria-related haemolytic condition, more clearly than they are now.

Cerebral Paragonimiasis With Hemorrhagic Stroke in a Developed Country.

Paragonimiasis is a food-borne parasitic disease caused by Paragonimus lung flukes, which are epidemic in Asia. Cerebral paragonimiasis accounts for <1% of symptomatic paragonimiasis but is the most common extrapulmonary infection. Cerebral paragonimiasis often mimics stroke and sometimes causes severe neurological sequelae. A 61-year-old woman was admitted to the hospital for severe headache. A head computed tomography scan revealed intracerebral hemorrhage with subarachnoid hemorrhage. The patient also had lesions in the lungs. She frequently ate Japanese mitten crab. Peripheral blood examination results of increased eosinophilia and immunological testing results confirmed the diagnosis of Paragonimus westermani infection. The patient was successfully treated with praziquantel as the first-line agent. Cerebral paragonimiasis is currently rare in developed countries; however, it is an important disease to consider.

Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads.

Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity.

Imported malaria in pregnant women experienced in Japan.

INTRODUCTION: With ever-growing global exchanges, the number of travelers, including pregnant women, to the tropics is increasing, which poses a risk of contracting malaria. Although there are several reports on imported malaria in pregnancy from Western countries, those focusing on cases experienced in Japan are very limited. METHODS: We searched for cases of malaria in pregnancy in the treatment records submitted to the Research Group on Chemotherapy of Tropical Diseases, Japan, during the period 1993-2016. Literature searches were also conducted using an American and a Japanese search system. RESULTS: Ten cases of malaria in pregnant women were identified, including four cases with Plasmodium falciparum. Of eight evaluable cases, only one practiced malaria chemoprophylaxis. Among the nine evaluable cases, eight resulted in uneventful delivery and one P. falciparum case developed severe hepatic disturbance, disseminated intravascular coagulation, and intrauterine fetal death. After the initial attack, none of the Plasmodium vivax/Plasmodium ovale cases practiced chloroquine prophylaxis until delivery. One P. ovale case received a lower dose regimen of chloroquine as acute-stage therapy. CONCLUSION: This study demonstrated additional cases of imported malaria in pregnant women to the literature and highlighted various epidemiological, demographic, and clinical characteristics. Some of the clinical issues raised need to be investigated. Due to the paucity of the cases worldwide, sharing information among various countries is indispensable, and international guidelines which are now increasingly recommending the use of artemisinins in pregnant women should be referred.

Thoracoscopic examination of empyema in a patient with sparganosis mansoni.

A 27-year-old man was admitted to our hospital with right pleural effusion. He had suffered from right chest and back pain and a high fever for one week prior to the admission. He had been treated with clarithromycin without improvement. Since thoracoscopy under local anesthesia revealed purulent effusion, synechiae and fibrous septa in the thoracic cavity, synechiotomy was performed and we started antibiotic treatment with the diagnosis of acute bacterial empyema. At the same time, we also suspected parasitic infection because of massive eosinophilic infiltration in pleural effusion and his dietary history of eating raw frogs. During the course of the disease, he had an infiltration in the right lower lobe and pneumothorax. Finally, we diagnosed him with sparganosis mansoni because his serum as well as pleural effusion was positive for the binding to sparganosis mansoni plerocercoid antigen, without any positive findings in bacteriology. His pleural effusion and lung infiltration were resolved after the administration of a high-dose praziquantel. We report this rare parasitic empyema with findings by thoracoscopic examination.

Optimal ELISA antigen for the diagnosis of Ascaris suum infection in humans.

Ascarid nematodes, Ascaris suum, Toxocara canis and Toxocara cati, are the most important causative species of larva migrans syndrome (LMS) in humans. Although the diagnosis of ascarid LMS is generally based on serological tests, specific serological tests for A. suum infection have not been fully developed. In the present study, the sensitivity and specificity of three A. suum antigen preparations, i.e., the somatic adult worm antigen (As-SWAP), larval excretory-secretory (ES) antigens derived from infective L3 (AsiL3-ES) and larval ES from tissue migratory L3 (AsmL3-ES), were evaluated for the serodiagnosis of A. suum infection in enzyme-linked immunosorbent assay (ELISA). We found that all A. suum antigen preparations showed positive reaction to all sera from A. suum-infected mice, while only AsmL3-ES obtained 100 % detection of anti-A. suum antibodies in human visceral ascarosis patients. Comparing the reactivity of each A. suum antigen, sera from both A. suum-infected mice and human patients bound to AsiL3-ES significantly weaker than As-SWAP and AsmL3-ES. Moreover, the OD450 values of ELISA with the A. suum antigen preparations and T. canis larval ES antigen (TciL3-ES) were compared in order to discriminate between ascarosis and toxocarosis. Linear discriminant analysis showed that diagnosis based on TciL3-ES and AsmL3-ES ELISA gave the most reliable result for the discrimination of infecting species. In conclusion, the application of AsmL3-ES antigen in ELISA can be recommended for the serodiagnosis of A. suum infection in humans.

Genome-wide variation in the pinewood nematode Bursaphelenchus xylophilus and its relationship with pathogenic traits.

BACKGROUND: Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease across Asia and Europe. In this study, we report the first genome-wide variation analysis of the nematode with an aim to obtain a full picture of its diversity. METHODS: We sequenced six key B. xylophilus strains using Illumina HiSeq sequencer. All the strains were isolated in Japan and have been widely used in previous studies. Detection of genomic variations were done by mapping the reads to the reference genome. RESULTS: Over 3 Mb of genetic variations, accounting for 4.1 % of the total genome, were detected as single nucleotide polymorphisms or small indels, suggesting multiple introductions of this invaded species from its native area into the country. The high level of genetic diversity of the pine wood nematode was related to its pathogenicity and ecological trait differences. Moreover, we identified a gene set affected by genomic variation, and functional annotation of those genes indicated that some of them had potential roles in pathogenesis. CONCLUSIONS: This study provides an important resource for understanding the population structure, pathogenicity and evolutionary ecology of the nematode, and further analysis based on this study with geographically diverse B. xylophilus populations will greatly accelerate our understanding of the complex evolutionary/epidemic history of this emerging pathogen.

Karyotype and reproduction mode of the rodent parasite Strongyloides venezuelensis.

SUMMARY Strongyloides venezuelensis is a parasitic nematode that infects rodents. Although Strongyloides species described to date are known to exhibit parthenogenetic reproduction in the parasitic stage of their life cycle and sexual reproduction in the free-living stage, we did not observe any free-living males in S. venezuelensis in our strain, suggesting that the nematode is likely to depend on parthenogenetic reproduction. We confirmed by cytological analysis that S. venezuelensis produces eggs by parthenogenesis during the parasitic stage of its life cycle. Phylogenetic analysis using nearly the full length of 18S and D3 region of 28S ribosomal RNA gene suggested that S. venezuelensis is distantly related to another rodent parasite, namely Strongyloides ratti, but more closely related to a ruminant parasite, Strongyloides papillosus. Karyotype analysis revealed S. venezuelensis reproduces with mitotic parthenogenesis, and has the same number of chromosomes as S. papillosus (2n = 4), but differs from S. ratti (2n = 6) in this regard. These results, taken together, suggest that S. venezuelensis evolved its parasitism for rodents independently from S. ratti and, therefore, is likely to have a different reproductive strategy.

ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

Notch2 signaling is required for proper mast cell distribution and mucosal immunity in the intestine.

Notch receptor-mediated signaling is involved in the developmental process and functional modulation of lymphocytes, as well as in mast cell differentiation. Here, we investigated whether Notch signaling is required for antipathogen host defense regulated by mast cells. Mast cells were rarely found in the small intestine of wild-type C57BL/6 mice but accumulated abnormally in the lamina propria of the small-intestinal mucosa of the Notch2-conditional knockout mice in naive status. When transplanted into mast cell-deficient W(sh)/W(sh) mice, Notch2-null bone marrow-derived mast cells were rarely found within the epithelial layer but abnormally localized to the lamina propria, whereas control bone marrow-derived mast cells were mainly found within the epithelial layer. After the infection of Notch2 knockout and control mice with L3 larvae of Strongyloides venezuelensis, the abundant number of mast cells was rapidly mobilized to the epithelial layer in the control mice. In contrast, mast cells were massively accumulated in the lamina propria of the small intestinal mucosa in Notch2-conditional knockout mice, accompanied by impaired eradication of Strongyloides venezuelensis. These findings indicate that cell-autonomous Notch2 signaling in mast cells is required for proper localization of intestinal mast cells and further imply a critical role of Notch signaling in the host-pathogen interface in the small intestine.

Pneumothorax with Eosinophilia is an Important Diagnostic Clue for Distinguishing Paragonimiasis from Chronic Eosinophilic Pneumonia: A Case Report.

The Paragonimus westermani infection is a parasitic foodborne infection that induces systemic symptoms with eosinophilia in humans. Here, we described pneumothorax in addition to pulmonary opacities with eosinophilia in a man with a positive P. westermani serology. He was misdiagnosed with chronic eosinophilic pneumonia (CEP) during the initial phase. Paragonimiasis can share similar clinical findings with CEP in cases where the worm is confined to the lungs. The findings of the current study suggest that paragonimiasis and CEP can be distinguished from each other by the presence of various symptoms. Notably, eosinophilia with pneumothorax should be an important diagnostic factor for paragonimiasis.

Population genetics study of Strongyloides fuelleborni and phylogenetic considerations on primate-infecting species of Strongyloides based on their mitochondrial genome sequences.

Strongyloides is a genus of parasitic nematodes of vertebrates comprising approximately 50 documented species, each with various host ranges. Among these, three species (S. stercoralis, S. fuelleborni, and S. cebus) are known to infect primate hosts. S. fuelleborni typically infects non-human primates in the Old World. To complement the existing information on the global genetic structure of this species, we conducted a genotyping study of S. fuelleborni samples collected from rhesus macaques in Myanmar, Japanese macaques in Japan, and some zoo-kept primates. This study identified a novel haplotype group in isolates from the Myanmar rhesus macaques. Subsequently, we obtained the complete or nearly complete mitochondrial genome sequences of S. fuelleborni, S. cebus (Strongyloides of New World monkeys), and S. vituli (Strongyloides of cattle). Phylogenetic analysis based on concatenated mitochondrial protein sequences of various Strongyloides species indicated a close relationship between S. fuelleborni, S. vituli and S. papillosus (Strongyloides in sheep and cattle). S. cebus is quite distantly related to both S. fuelleborni and S. stercoralis, which led to the hypothesis that the three primate Strongyloides species evolved independently as parasites of primates.