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Schistosomiasis remains a worldwide public health problem, especially in sub-Saharan Africa. The World Health Organization targets the goal for its elimination as a public health problem in the 2030 Neglected Tropical Diseases (NTDs) Roadmap. Concerted action and agile responses to challenges will be necessary to achieve the targets. Better diagnostic tests can accelerate progress towards the elimination by monitoring disease trends and evaluating the effectiveness of interventions; however, current examinations such as Kato-Katz technique are of limited power to detect light-intensity infections. The point-of-care circulating cathodic antigen (POC-CCA) test shows a higher sensitivity compared to the reference standard, Kato-Katz technique, but it still lacks sufficient sensitivity with low infection intensity. In this study, we examined antibody reactions against recombinant protein antigens; Schistosoma mansoni serine protease-inhibitor (SmSerpin) and RP26, by enzyme-linked immunosorbent assay (ELISA) in plasma samples with light-intensity infection. The sensitivity using the cocktail antigen of recombinant SmSerpin and RP26 showed 83.7%. The sensitivity using S. mansoni soluble egg antigen (SmSEA) was 90.8%, but it showed poor specificity (29.7%), while the cocktail antigen presented improved specificity (61.4%). We conclude that antibody detection to the SmSerpin and RP26 protein antigens is effective to detect S. mansoni light-intensity infections. Our study indicates the potential of detecting antibody against recombinant protein antigens to monitor the transmission of schistosomiasis in low endemicity contexts.

Background: Larva migrans syndrome is a food-borne parasitic disease in humans, caused by accidental ingestion of eggs or larvae of ascarid nematodes, namely, Toxocara canis, Toxocara cati, or Ascaris suum, the roundworms commonly found in the intestines of dogs, cats and pigs respectively. When a patient is diagnosed as having larva migrans syndrome, oral-administration of albendazole is recommended, however, the regimen remains controversial worldwide. In Japan, the duration of albendazole administration is longer than those of European and North American countries. The purpose of this study was to assess the efficacy and safety of long-term administration treatment of albendazole for larva migrans syndrome. Methods: From 2004 to 2014, our laboratory was involved in the diagnosis of 758 larva migrans syndrome cases, of which 299 cases could be followed up after the treatment. We analyzed these 299 follow-up cases on the ELISA results before and after the treatment as well as on anthelmintic used, dose and duration of medication, clinical findings, and side effects, recorded on a consultation sheet provided by the attending physicians. We have 288 cases as the subjects of this study. Results: Albendazole represented a 78.0% efficacy rate. The side effects represented 15.0% in using albendazole alone cases; however, the side effects were mild to moderate and there were no severe cases reported. Conclusions: The long-term administration treatment of albendazole is safe and effective for larva migrans syndrome. Keywords: Albendazole, Larva migrans syndrome, Ascaris suum, Toxocara canis, Toxocara cati

Objective Visceral larva migrans (VLM) caused by Ascaris suum is a major health problem in pig farming regions. The clinical characteristics of pulmonary VLM caused by A. suum, however, are unclear. We assessed the clinico-radiologic features of this disease. Methods Medical records, including the results of chest radiography and high-resolution computed tomography (HRCT), were retrospectively reviewed from January 2000 through June 2019, at the University of Miyazaki Hospital and Kyoritsuiin Hospital in Miyazaki Prefecture, Japan. Results Seven patients with VLM caused by A. suum were identified. All seven patients had a unique habit of consuming raw foods, such as organic vegetables, chicken, turkey, wild boar, and venison. All but one patient, who had eosinophilic pneumonia with a fever and severe fatigue, had only mild or no respiratory symptoms. All 7 patients had remarkable eosinophilia (median, 1,960 /μL) and high serum IgE levels (median, 1,346 IU/mL). Chest HRCT revealed multiple nodules and multiple nodular ground-glass opacities in 57% and 29% of the patients, respectively. The pulmonary lesions were located predominantly in subpleural areas. All seven patients were treated with albendazole, which led to improvement within two to three months. Neither eggs nor parasites were detected in the feces or sputum of any patient. Conclusion Consumption of raw organic vegetables or raw meat is a possible route of A. suum infection. Infected patients exhibit mild respiratory symptoms, and multiple nodules with a halo in the subpleural area are a common finding on chest HRCT. Treatment with albendazole was effective in these cases.

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.

Background Eosinophilic meningitis (EM) is a rare clinical syndrome caused by both infectious and noninfectious diseases. In tropical pacific countries, Angiostrongylus cantonensis is the most common cause. However, the EM definition varies in the literature, and its relation to parasitic meningitis (PM) remains unclear. Methodology/Principal findings Adult and adolescent patients of 13 years old or above with suspected central nervous system (CNS) infections with abnormal CSF findings were prospectively enrolled at a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. Patients with EM or suspected PM (EM/PM) were defined by the presence of either ≥10% eosinophils or an absolute eosinophil cell counts of ≥10/mm3 in the CSF or blood eosinophilia (>16% of WBCs) without CSF eosinophils. In total 679 patients were enrolled: 7 (1.03%) had ≥10% CSF eosinophilia, 20 (2.95%) had ≥10/mm3 CSF eosinophilia, and 7 (1.03%) had >16% blood eosinophilia. The patients with ≥10% CSF eosinophilia were significantly younger (p = 0.017), had a lower body temperature (p = 0.036) than patients with ≥10/mm3 CSF eosinophilia among whom bacterial pathogens were detected in 72.2% (13/18) of those who were tested by culture and/or PCR. In contrast, the characteristics of the patients with >16% blood eosinophilia resembled those of patients with ≥10% CSF eosinophilia. We further conducted serological tests and real-time PCR to identify A. cantonensis. Serology or real-time PCR was positive in 3 (42.8%) patients with ≥10% CSF eosinophilia and 6 (85.7%) patients with >16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia. Conclusions The etiology of PM in northern Vietnam is A. cantonensis. The eosinophil percentage is a more reliable predictor of parasitic EM than absolute eosinophil count in the CSF. Patients with PM may present with a high percentage of eosinophils in the peripheral blood but not in the CSF., Author summary Eosinophilic meningitis (EM) is a rare meningitis accompanied by eosinophils in the CSF and caused by multiple etiologies. Angiostrongylus cantonensis, which is a rat lungworm parasite, is the most common cause in tropical Asia. Previous papers have defined EM as CSF eosinophils ≥10% or CSF eosinophils ≥10/mm3. However, the relationship of EM to parasitic meningitis (PM) remains unclear. This prospective study enrolled 679 patients with suspected CNS infection who were admitted to a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. The characteristics of patients with ≥10% CSF eosinophilia resembled those of patients with >16% blood eosinophilia without CSF eosinophils, whereas those of patients with ≥10/mm3 CSF eosinophilia were comparable with those of patients with typical bacterial meningitis. Serology or real-time PCR for A. cantonensis was positive in 3 out of 7 patients with ≥10% CSF eosinophilia and 6 out of 7 patients with > 16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia. The percentage, in contrast to the absolute eosinophil count in CSF, is reliable for predicting parasitic EM. Patients with PM may present with eosinophilia in the peripheral blood but not in the CSF.

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.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum’s phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism., Kikuchi et al. sequence the genome of the fatal tapeworm Sparganum proliferum and a closely related non–life-threatening tapeworm Spirometra erinaceieuropaei, and describe its genomic features suggesting the natural history and molecular mechanisms underlying pathogenicity. Their findings indicate that nonordinal extracellular matrix coordination is important for its asexual reproduction, and suggest that loss of sexual maturity contributes to the fatal pathogenicity of S. proliferum to humans.

Fascioliasis is a parasitic infestation caused by the digenetic trematodes Fasciola hepatica and F. gigantica. It is not commonly seen in developed countries, so diagnosis there is always difficult as a result of confusion with other hepatic or biliary disorders. A 56-year-old man presented at our hospital with a hepatic mass that had been inadvertently discovered by ultrasonography. Abdominal computed tomography revealed a multi-cystic lesion distributed along the branch of the right bile duct. Endoscopic retrograde cholangiopancreatography showed serrated changes ranging from the upper level of the common bile duct to the right hepatic bile duct. Eosinophilia was not observed and tumor marker levels were within normal ranges. Following right lobectomy combined with bile duct reconstruction, a histological examination revealed cholangitis with inflammatory cell infiltration accompanied by parasite egg-like structures and Charcot-Leyden crystals. An additional serologic test was positive for F. hepatica antibodies. A diagnosis of fascioliasis was thus confirmed by histopathology and serology. Fascioliasis should be suspected if imaging findings such as multiple small hypodense lesions in the liver are observed, and serologic tests can be useful for differential diagnosis.

Development of reliable, quantitative technologies for serodiagnosis of Toxoplasma gondii infection remains desirable. The luciferase immunoprecipitation system (LIPS) is a relatively simple, highly sensitive, and rapid quantitative immunoassay. The major advantages of this assay over ELISA are a wider dynamic range, shorter overall assay time, and less sample volume. In this study, we aimed to use this method for the serodiagnosis of toxoplasmosis. Recombinant Toxoplasma antigens (dense granule antigens GRA6, GRA7, and GRA8 and bradyzoite antigen BAG1) fused with nanoluciferase (Nluc, a small luciferase enzyme) were expressed in Escherichia coli, purified, and tested in LIPS assays with sera from experimental mice infected with T. gondii and a WHO standard anti-Toxoplasma human immunoglobulin (TOXM). In the experimentally infected mice, LIPS assays detected antibodies against Nluc-GRA6, Nluc-GRA7, and Nluc-GRA8 as early as day 14, whereas antibodies against Nluc-BAG1 remained undetected until day 21 and then showed significant elevation on day 60. In TOXM sera, LIPS assays with each Nluc recombinant protein produced reliable standard curves with a coefficient of determination (R2) of 0.980–0.989 for GRA6, 0.986–0.990 for GRA7, 0.998–0.999 for GRA8, and 0.942–0.987 for BAG1. The detection limits were estimated to be 3.9, 2, 1, and 1 IU/ml for rGRA6, rGRA7, rGRA8, and rBAG1, respectively. The LIPS assay for toxoplasmosis could detect antibodies against T. gondii in the mouse and human sera with a reasonably high sensitivity. We consider the LIPS assay to be a promising alternative tool for screening, diagnosing, and monitoring toxoplasmosis. In particular, detection of antibodies against BAG1 may be useful for a longitudinal seroprevalence study in suspected high-risk areas on the basis of its elevated serum concentration in the chronic phase.

The feline F12 gene was examined to identify a mutation associated with coagulation factor XII (FXII) deficiency in a litter of 6 cats, including 2 cats with severely reduced FXII activity (7.1 and 9.3%, respectively) and 4 cats with moderately reduced FXII activity (range 36.0 to 46.3%). Cats with severely reduced FXII activity were homozygous for a G to C missense mutation in exon 13 of the F12 gene, resulting in an amino acid change (p.G544A). Cats with moderately reduced FXII activity were heterozygous for this mutation. Expression studies revealed reduced secretion of p.G544A mutant FXII protein from transfected HEK293 cells compared with wild type FXII. These results reveal a novel F12 mutation in FXII deficient cats and define the underlying mechanism for low FXII activity in homozygotes.

Proliferative sparganosis is one of the most bizarre and mysterious parasitic diseases ever described. The causative parasite is Sparganum proliferum, which is a pseudophyllidean cestode distinct from Spirometra tapeworms. Here we overview this rare but fascinating disease with the all original case reports on human patients published in the last 115 years. Proliferative sparganosis is clearly divided into two disease types, cutaneous and internal proliferative sparganosis. Cutaneous type starts with a skin eruption caused by the dermal invasion of a sparganum. Skin lesion progresses to larger areas of the body if left untreated. Various internal organs and body wall can be eventually affected. The clinical symptoms of patients in this group are very similar to each other. Molecular data suggest that cutaneous proliferative sparganosis is caused by S. proliferum of which genetic variation is limited, regardless of the time or localities of the emergence of patients. Internal proliferative sparganosis, on the other hand, is much more heterogeneous. Some cases show aggressive infection in internal organs, while others show only restricted lesions. Some of the cases that had been cited as proliferative sparganosis in the past literature were removed from the list, because they were judged as cyclophyllidean tapeworm infections. DNA sequencing is mandatory for the definite diagnosis of proliferative sparganosis. The Venezuelan strain of S. proliferum is maintained in experimental mice in Japan, which is fully prepared for the experimental study with advanced technologies in modern molecular biology.

The receptor for advanced glycation end products (RAGE) recognizes Ca++-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca++-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca++ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca++-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes.

Paragonimiasis, or lung fluke disease, is a typical food-borne parasitic zoonosis caused by infection with trematodes belonging to the genus Paragonimus. More than 50 species of Paragonimus have been reported throughout the world, of which seven valid species infect humans, an estimated one million people annually worldwide. Among the seven species, P. westermani, P. heterotremus, and P. skrjabini/P. s. miyazakii, distributed in Asia, are the most important species as the cause of paragonimiasis. Humans acquire infection through the ingestion of raw, pickled or undercooked freshwater crustaceans, 2nd intermediate hosts, or consuming raw meat of wild boar or deer, paratenic hosts. Infections often occur clustered in foci where dietary habits allow transmission of the parasites. Paragonimiasis typically causes a subacute to chronic inflammatory disease of the lungs. The symptoms, including chronic cough, chest pain, dyspnea and hemoptysis, mimic those of tuberculosis and lung cancer. Serologic tests are commonly used for the diagnosis of paragonimiasis, and Praziquantel is the treatment of choice. In this review, the current status of Paragonimus and paragonimiasis in Asia is outlined based on the latest information and findings. We also summarize current trends of paragonimiasis in Japan, which is one of the most endemic area of paragonimiasis in the world, for the better understanding and control of paragonimiasis.

Cutaneous gnathostomiasis (CG) is a disease caused by ingestion of third-stage Gnathostoma larva in raw snakes, freshwater fish or frogs. The common causative organisms of CG in Japan include G. nipponicum, G. spinigerum, G. doloresi, G. binucleatum and G. hispidum. We report two cases of CG after eating many raw Japanese icefishes (Salangichthys microdon). In both cases, linear itchy eruptions on the trunk developed after eating many S. microdon. We performed genetic analysis in the first case, which revealed G. nipponicum. Of note, this is the first case of CG diagnosed based on genetic analysis in Japan. In Japan, eating whole small raw freshwater fish is common. The most popular types of raw small freshwater fish consumed in Japan are S. microdon (shirauo in Japanese) and Leucopsarion petersii (shirouo in Japanese). Usually, S. microdon are born in rivers, but live in both the sea and rivers. They feed on small fish and freshwater water fleas and spawn in rivers in the spring. On the other hand, L. petersii are born in rivers, but move to the sea soon after hatching. They feed on plankton such as copepod in the sea. They do not feed on anything when they return to rivers to spawn in the spring. Therefore, we hypothesize that S. microdon are more easily parasitized by G. nipponicum.

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.

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.

An 11-year-old boy living in Otsu City, Shiga Prefecture, Kansai Region, Western Honshu, Japan had zoonotic onchocercosis. The patient developed a painful swelling on the little finger of his left hand. The worm detected in the excised mass had external transverse ridges but did not have inner striae in the cuticle. On the basis of the parasite's histopathological characteristics, the causative agent was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species of the filarial parasite was confirmed by sequencing the cox1 gene of the parasite. The Japanese wild boar Sus scrofa leucomystax is a definitive host for O. dewittei japonica, which is then transmitted by blackflies as the vector to humans. The current case described occurred in the Kansai Region, Western Honshu, where such infections were previously not reported.

Paragonimiasis is a food-borne parasitic disease caused by Paragonimus lung flukes, which are epidemic in Asia. Cerebral paragonimiasis accounts for

Toxocara canis is a parasite known to cause visceral larva migrans. The infection rarely affects the central nervous system but there have been several reports of myelitis caused by visceral larva migrans due to Toxocara canis. In previous reported cases, the lesions were located in the thoracic or cervical spinal cord. To the best of our knowledge, this is the first report of a lesion involving the lumbosacral region.A 60-year-old man developed weakness and dysesthesia in the lower limbs. The symptoms resolved spontaneously, but recurred after five months. One month later, the patient developed pollakiuria and constipation. He was a dog owner and frequently ate raw chicken meat and beef liver. Sagittal T2-weighted image (T2WI) showed swelling and hyperintensity in the spinal cord from T10 to the lumbosacral region and focal nodular enhancement on the posterior segment of the lumbar spinal cord. Blood cell counts showed slight eosinophilia and elevated serum immunoglobulin E level. Cerebrospinal fluid examination showed slight pleocytosis with eosinophilia. Enzyme-linked immunosorbent assay showed high levels of anti-Toxocara antibodies in the serum and cerebrospinal fluid. In addition, confirmatory test by Western blot was positive. The patient was initially treated with intravenous methylprednisolone with slight improvement in muscle weakness. Albendazole was added with a second course of intravenous methylprednisolone. The muscle weakness in the lower limbs improved considerably, and swelling and hyperintensity on T2WI almost disappeared.Our results suggest that Toxocara canis myelitis cannot be discounted even if the myelitis involves the lumbosacral region.

Summary Background Recently, a dose of 30 mg (base) primaquine daily for 14 days is increasingly recommended for radical cure of Plasmodium vivax malaria. However, total primaquine doses, or those per body weight, are also recognized as important. In Japan, primaquine is not a licensed medicine, but has been used through the Research Group on Chemotherapy of Tropical Diseases for >3 decades. Methods Based on clinical records submitted to the Research Group, patients with P. vivax and Plasmodium ovale malaria treated with primaquine were analyzed to determine the efficacy and safety of the antimalarial drug. Results Seventy-five P. vivax cases, including 3 in children, and 19 P. ovale cases were enrolled. Five of the P. vivax cases demonstrated at least one relapse despite primaquine therapy. Total primaquine doses per body weight were obtained in 4 of the 5 relapsed patients, presenting 9 malaria episodes totally, and most of the primaquine failures were caused with a total dose ≤3.5 mg/kg. Liver function disturbance was reported in 2 cases. Conclusion In order to optimize radical cure of P. vivax, the total primaquine dose per body weight should be considered, at least 3.5 mg/kg or even more if contracted in countries with significant drug resistance. Possibility of primaquine hepatotoxicity in chronic liver disease patients remains to be elucidated.

Objective Paragonimiasis, or lung fluke infection, is a food-borne parasitic disease caused by infection with trematodes belonging to the genus Paragonimus. Although paragonimiasis was once considered successfully controlled in the 1970s, new cases began to emerge in the late 1980s. To apprehend the current-day situation of the re-emergent cases of paragonimiasis in Japan, we conducted a retrospective review of 443 patients who were referred to our laboratory and diagnosed as having paragonimiasis during 2001-2012. Methods Patients were diagnosed as having paragonimiasis based primarily on immunodiagnostic methods in addition to clinical, laboratory, and radiographic findings. Patient data were extracted from consultation sheets from attending physicians and were analyzed. Results Majority of the patients were residents of Kyushu Island. However, a substantial number of cases were also from other parts of Japan. Immigrants (mostly from China, Thailand, and Korea) accounted for a quarter of the cases. Native Japanese contracted paragonimiasis by consuming wild boar meat or freshwater crabs, whereas immigrants contracted the infection almost exclusively by consumption of freshwater crabs. Eosinophilia and elevated serum IgE levels were found in around 80% of the patients. Parasite egg detection was documented only in 11.7% of the cases, showing the reliance on serological tests for diagnosing paragonimiasis in current clinical practice. Conclusion Paragonimiasis remains a public health issue in Japan, and the situation should be closely monitored.

Background The Research Group on Chemotherapy of Tropical Diseases, Japan, introduced artemether–lumefantrine (AL) in late 2002, mainly for treating uncomplicated Plasmodium falciparum malaria. Because AL was on the market in Japan in March 2017, the effectiveness and safety of AL were analyzed to help medical personnel use AL optimally. Methods Case report forms submitted by the attending physicians were analyzed. When necessary, direct contact with the attending physicians was made to obtain detailed information. Results Effectiveness analysis was performed for 62 cases and safety analysis was performed for 66 cases. In P. falciparum malaria, the overall cure rate was 91.1% (51/56), of which the cure rates for Japanese and non-Japanese patients were 82.1% (23/28) and 100% (28/28), respectively. The successfully treated cases included severe P. falciparum malaria, with parasite densities exceeding 500,000/μL. Adverse events were reported in 14 patients, including delayed hemolytic anemia which occurred in the top four highest parasitemic cases. Conclusions AL treatment failure in P. falciparum malaria may not be rare among non-immune individuals, including Japanese. The possibility of delayed hemolytic anemia, which occurs preferentially in high parasitemic cases, should be considered following AL treatment.

An 11-year-old boy collapsed during morning assembly at his junior high school. The automated external defibrillator detected ventricular fibrillation and provided shock delivery. He was successfully resuscitated and reverted to sinus rhythm. Electrocardiography showed ST-T elevation in the precordial leads. Echocardiography and angiography demonstrated akinesia of the apex and mid-wall of the left ventricle with preserved contraction of the basal segments, which suggested Takotsubo cardiomyopathy. The patient and his family had often eaten uncooked crab, and his father had a past history of infection with Paragonimiasis westermani. The patient had had a persistent cough and chest pain for several weeks. Chest radiograph showed cystic cavities in the left upper lung. Microbiological examination of the sputum demonstrated an egg of P. westermani and immunological assay showed a raised antibody titre to P. westermani. On the12th day of admission, he developed seizures, and magnetic resonance imaging demonstrated cerebral involvement. After the administration of praziquantel for 3 days, the clinical manifestations improved immediately, and echocardiography normalised within 3 weeks. The patient was discharged on the 32nd day + and follow-up was normal. Takotsubo cardiomyopathy following a potentially fatal arrhythmia is a rare cardiac complication associated with pulmonary and central nervous system infection by P. westermani.

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.

Humans and dogs are the two major hosts of Strongyloides stercoralis, an intestinal parasitic nematode. To better understand the phylogenetic relationships among S. stercoralis isolates infecting humans and dogs and to assess the zoonotic potential of this parasite, we analyzed mitochondrial Cox1, nuclear 18S rDNA, 28S rDNA, and a major sperm protein domain-containing protein genes. Overall, our analyses indicated the presence of two distinct lineages of S. stercoralis (referred to as type A and type B). While type A parasites were isolated both from humans and dogs in different countries, type B parasites were found exclusively in dogs, indicating that the type B has not adapted to infect humans. These epidemiological data, together with the close phylogenetic relationship of S. stercoralis with S. procyonis, a Strongyloides parasite of raccoons, possibly indicates that S. stercoralis originally evolved as a canid parasite, and later spread into humans. The inability to infect humans might be an ancestral character of this species and the type B might be surmised to be an origin population from which human-infecting strains are derived.

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.

Recently, reports of delayed hemolytic anemia after treatment with artemisinin and its derivatives have emerged. Here we report two cases of delayed hemolytic anemia in a patient with severe falciparum malaria after treatment with oral artemether-lumefantrine (AL). The first patient, a 20-year-old Japanese male student, was diagnosed with falciparum malaria and was administered AL. As having a high parasitemia rate (20.6%) was the only severe malaria criterion met in this case and his general condition was stable, we continued with AL treatment. Despite disappearance of malarial parasites after 4 days of AL administration, a persistent fever remained. On days 13 and 16, a diagnosis of hemolytic anemia was made (lactate dehydrogenase [LDH]: 1,466 U/L, hemoglobin [Hb]: 7.2 g/dL). A blood smear at that time revealed no parasites. He recovered naturally from delayed hemolysis. The second patient, a 27-year-old Japanese female student, was diagnosed with falciparum malaria (parasitemia: 4.5%) and treated initially with oral quinine hydrochloride and doxycycline. The following day, parasitemia increased to 7.9% and oral AL was initiated. She was discharged on day 4 after achieving parasite clearance and afebrility. However, on day 5, fever (body temperature > 38°C) recurred, and on day 11, a diagnosis of hemolytic anemia was made (LDH: 712 U/L, Hb: 8.8 g/dL). A follow-up confirmed that her condition improved gradually. AL treatment of severe malaria can cause delayed hemolytic anemia. Patients should be followed up for up to 4 weeks to detect signs of hemolysis and provide appropriate symptomatic treatment.

A 53-year-old woman was admitted to the department of neurology in Tenri Hospital because of progressive thoracic myelitis a month after she had eaten uncooked bovine liver. A previous episode of right optic neuritis and a positive test for serum anti-aquaporin-4 antibodies indicated a diagnosis of neuromyelitis optica spectrum disorders. Although the patient initially recovered with the reduction of anti-aquaporin-4 antibodies during treatment with intravenous methylprednisolone infusion and plasma exchange, her neurological symptoms deteriorated soon after the completion of plasma exchange. Western blotting analysis detected anti-Toxocara canis antibodies in the serum; thus, the patient underwent oral albendazole treatment. This resulted in the alleviation of her symptoms. We therefore consider that rigorous investigation should be encouraged to detect rare pathogens including parasites in cases of treatment-resistant neuromyelitis optica spectrum disorders.

A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0–12.4%, and interassay CV was 11.5–12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs ( P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.

A disintegrin and metalloproteinase with thrombospondin type 1 motifs, number 13 (ADAMTS13) is a plasma zinc metalloprotease also known as von Willebrand factor (VWF)-cleaving protease. Deficiency of ADAMTS13 activity is known to cause thrombotic thrombocytopenic purpura (TTP) in humans. We isolated the canine ADAMTS13 cDNA, which encodes 1502 amino acids, and expressed the recombinant protein to evaluate VWF-cleaving ability. Although the propeptide domain was longer and the TSP1 repeat domain was shorter than those in other species, the overall structures were similar to human and mouse ADAMTS13. Recombinant canine ADAMTS13 cleaved the 250-kDa VWF monomer into two fragments of 150 kDa and 120 kDa. Furthermore, high molecular weight VWF multimers were abolished based on the activity of ADAMTS13. These results could facilitate research into hemostatic disorders such as TTP in dogs.

Background Eosinophilic myocarditis is a rare clinical entity characterized by eosinophilia and myocardial inflammation with infiltrating eosinophils. The prognosis of patients with eosinophilic myocarditis is difficult to determine due the disease's rarity and varied causes; consequently, standard treatment has not been established. Objective To elucidate the clinical characteristics and treatment outcome of eosinophilic myocarditis, we retrospectively studied 7 patients fulfilling the criteria of the Japanese Circulation Society for eosinophilic myocarditis from among 64 patients admitted to our institution with eosinophilia over a 27-year period. Results The patients' ages at diagnosis ranged from 36 to 83 years (median: 52 years). The etiologies of the eosinophilic myocarditis were found to be idiopathic (3 patients), Churg-Strauss syndrome (2 patients), parasitic infection (1 patient) and chronic eosinophilic leukemia (CEL) (1 patient). In addition to treatment for the underlying disease, we also administered prednisolone at a dose appropriate to the disease severity (6 of 7 patients). The patient who was diagnosed with a parasitic infection was treated only with albendazole, because eosinophilic myocarditis was mild. The patient with CEL was positive for the FIP1 L1-PDGFRα fusion gene and was treated with imatinib. Eosinophilic cationic protein was a useful marker for assessing disease activity and treatment efficacy. At the end of the study, of the seven patients treated, six were alive (86%), giving a mean survival time of 37 ± 40 months (mean ± SD). Conclusion Because eosinophilic myocarditis has various etiologies, it is essential to identify the etiology of the underlying disease. In the majority of eosinophilic myocarditis patients, administration of prednisolone may be an effective therapeutic modality producing a good outcome.

We report a case of creeping disease treated successfully with ivermectin. A 46-year-old man presented with a 1-month history of pruriginous linear erythema on his right thigh after a visit to Indonesia. Although he had no history of eating raw fish or meat, he walked along the river and in the jungle without wearing shoes. Creeping disease caused by animal hookworm was strongly suspected. The presence of parasite larvae was not confirmed in biopsied skin specimens. In enzyme-linked immunosorbent assay, serum samples were negative for binding to hookworm antigens, including Ancylostoma canium, Necator americanus and Gnathostoma doloresi. He was treated with a single 12 mg oral dose (200 microg/kg) of ivermectin. The eruption and pruritus resolved within a few days after the administration and did not relapse.

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.

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

Anti-idiotypic antibodies (Ab2) mimicking antigens (Ags)-defined by antibodies (Ab1) directed to tumors or pathogens have elicited Ag-specific humoral, cellular and/or protective immunity in experimental animals and in humans. In immunizations of rodents with Ab1, factors such as animal species, form of Ab1 and choice of adjuvant are crucial for the successful induction of Ab2 as candidate vaccines against tumors and pathogens. Here we survey the outcome of 362 fusion events (each event representing one animal), using nine immunization schedules in mice and seven schedules in rats and including 10 different Ab1 directed against human tumor- and immunodeficiency virus (HIV-1)-associated Ags. Ab1 IgG or F(ab′) 2 were administered uncoupled or coupled to keyhole limpet hemocyanin (KLH). As adjuvants, complete and incomplete Freund's adjuvant (CFA/IFA), lipid A, aluminum hydroxide, TiterMax or vaccinia virus were used. In syngeneic immunizations with murine Ab1 in mice, F(ab′) 2 coupled to KLH and emulsified in CFA/IFA preferentially induced Ab2 mimicking tumor or HIV-1 associated epitopes. In xenogeneic immunizations with mouse Ab1 in rats, various forms of Ab1 and adjuvants successfully induced Ab2 mimicking tumor Ags.

One of the most common zoonotic helminth infections is caused by species in the genus Toxocara, particularly Toxocara canis and T. cati (Syn. T. mystax). However, their relative contribution to toxocarosis in humans remains largely unknown because causative larvae are seldom recovered and uncertainties regarding the validity of existing serological assays. In this study, we used sera from a pig model experimentally infected with T. canis and T. cati to evaluate whether a Western blot could discriminate between the two species. No proteins were observed that could be used as a diagnostic tool. In addition, a heterogenic protein pattern between individual hosts was found, which was most pronounced in the T. cati-infected pigs. There is therefore an urgent need to optimize and validate current methods or develop new species-specific serological methods in order to implement appropriate control measures.

Introduction Paragonimiasis is a food-borne infection caused by Paragonimus parasites. The lungs and pleura are the primary sites for the infection; however, ectopic infection can occur in other organs such as skin, liver and brain. It is difficult to make a diagnosis of ectopic paragonimiasis due to an ignorance of, and unfamiliarity with the disease. We report the case of a patient with subcutaneous paragonimiasis diagnosed by histopathological analysis and serological testing. Case presentation A 39-year-old Chinese immigrant woman presented with a subcutaneous nodule in her left lower back. The nodule was initially suspected of lipoma and she was followed up on without any treatment. However, it gradually indurated and the nodule was resected surgically. A magnetic resonance imaging scan revealed a polycystic lesion with inhomogeneous low or high intensity on T1- or T2-weighted images, respectively. The rim of the lesion was enhanced after contrast enhancement, but the inside did not show high-signal intensity. A histological analysis of the surgically resected specimen revealed variable-sized tubulo-cystic structures. The cyst wall showed a granulomatous change with scant eosinophilic infiltration. A number of parasite ova were observed in the necrotic tissue inside the cysts, and a parasite body with a presumed oral sucker and reproductive organ was also detected, suggesting a trematode infection. A subsequent serological examination showed a positive reaction of her serum to the Paragonimus westermani antigen. No abnormal findings were found on her chest computed tomography scan. The diagnosis of subcutaneous paragonimiasis caused by Paragonimus westermani was made. Conclusions We report a case presenting only as a non-migratory subcutaneous nodule without any pleuropulmonary lesion, which was initially suspected of lipoma but denied by magnetic resonance imaging scan results. The case was subsequently diagnosed as subcutaneous paragonimiasis from the results of histopathological analysis and serological testing.

Background: Zoonotic infections with Onchocerca species are uncommon, and to date only 25 clinical cases have been reported worldwide. In Japan, five previous zoonotic infections were concentrated in Oita, Kyushu (the southern island), with one previous case in Hiroshima in the western part of Honshu (the main island). The causative agent in Japan was identified as Onchocerca dewittei japonica Uni, Bain & Takaoka, 2001 from Japanese wild boars (Sus scrofa leucomystax Temminck, 1842). Here we report two infections caused by a female and male O. dewittei japonica, respectively, among residents of Hiroshima and Shimane Prefectures in the western part of Honshu. Methods: In both cases, nodules were surgically removed. The parasites in nodules were identified on the basis of their histopathological characteristics. Identification was confirmed by sequencing the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from worms in the tissues used in the histological preparations. Results: Case 1 was a 61-year-old woman from Hiroshima Prefecture who complained of a painful subcutaneous nodule on the back of her right hand. The causative agent was identified as a female O. dewittei japonica owing to transverse ridges on the cuticle and molecular analysis. Case 2 was a 78-year-old woman from Shimane Prefecture who had a painful nodule in the left temporal region. Histopathological characteristics and cox1 sequencing of the worm indicated that the causative agent was a male O. dewittei japonica. Conclusions: For Cases 1 and 2, we diagnosed the causative agents as a female and male O. dewittei japonica, respectively. These findings indicate the spread of a zoonosis caused by O. dewittei japonica in the western part of Honshu, where wild boars have recently extended their habitats because of decreased annual snowfall, unused rice fields and a decline in the number of hunters in Japan. The O. dewittei japonica infection rate among wild boars was reported as 78% in Shimane Prefecture, in the western part of Honshu. Therefore, in the near future, zoonotic onchocercosis is likely to occur in Honshu as well as Kyushu, where wild boars, blackfly vectors and humans share the same habitat.

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector.

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

We recently reported that the rat mast cell proteinase inhibitor trypstatin is genetically identical with the second half of inter-alpha-trypsin inhibitor light chain (ITI-LC), also known as bikunin or urinary trypsin inhibitor (UTI). In this study, therefore, immunoreactivities of mast cells of various human tissues were examined with three antibodies, anti-human ITI-LC, anti-ITI, which recognizes mainly heavy chains or the sugar moiety of ITI, and anti-alpha 1-microglobulin (alpha1mG). ITI-LC immunoreactivity was strongly found in mast cells in the connective tissues of various organs except for those of the propria mucosae of small intestine. Neither anti-ITI antibody nor anti-alpha1mG antibody reacted with mast cells in various tissues. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, alpha1mG/ITI-LC mRNA was not detected in the skin and tongue, and only weakly in small intestine, although ITI-LC immunoreactivity was strongly detected in these tissues. Furthermore, the mRNA was not expressed in cultured human mast cells. These results suggest that ITI-LC protein is stored in the granules of human connective tissue mast cells, though is not produced by them.

To establish a screening procedure for tumor cell-surface reactive Fabs, we used a model antigen/antibody system including the epidermal growth factor receptor (EGF-R) and the anti-EGF-R monoclonal antibody 425. The 425 Fab was displayed on the surface of M13 filamentous phage. In a screening assay for 425 phage binding to tumor cell surfaces, biotinylated 425-phage bound specifically to EGF-R-positive A431 epidermoid carcinoma cells and not to K562 non-expressor erythroleukemia cells. With a model library, the sensitivity of phage enrichment by phage binding to cell surfaces was one 425-phage in 20,000 unrelated phages after 4 rounds of panning on A431 cells. In a phage tissue screening assay, 425-phage, but not unrelated phage, bound specifically to melanoma cells expressing EGF-R. Epitope and idiotope specificity of 425-phage was demonstrated in phage competition assays, using as targets A431 cells and anti-idiotypic antibodies to monoclonal antibody 425, respectively. Finally, the EGF-R protein was directly isolated from A431 cell extracts, using biotinylated 425-phage. The data obtained with the 425 model library system demonstrate the usefulness of antibody phage display for the rapid identification and isolation of tumor or other disease-related cell surface antigens.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma antigens in patients. We have attempted to identify melanoma-associated antigens recognized by patients' B cells using an antibody phage display approach. Antibody display on filamentous phages allows direct screening of cDNA libraries for expression of cell-surface-reactive antibodies, without the need for antibody production and purification using bacteria or eukaryotic cell systems. This approach was used to identify melanoma-associated cell-surface antigens recognized by patients' B cells. Antibodies produced by the B cells of a melanoma patient (in remission for > 7 years following periodic vaccination with allogeneic melanoma cell vaccine) were displayed as Fabs on the surfaces of filamentous phages. A library of 10(8) phages was absorbed to normal melanocytes, followed by phage binding to and elution from melanoma cells (human lymphocyte antigen nonmatched and vaccine melanoma cells). Phages were further selected for reactivities with tunicamycin-treated melanoma cells. These procedures resulted in a > 10(6)-fold enrichment of tumor-specific phages from the original phage library. One phage-Fab bound to melanoma cells, other tumor cells, and a few normal cells in cultured cell lines and in tissue sections.

The clinical management of amebiasis is a growing concern, particularly among human immunodeficiency virus (HIV)-infected individuals who are predisposed to severe illness. Treatment with a luminal amebicide is strongly recommended following acute-stage treatment with a nitroimidazole. In 2004, the Japanese Research Group on Chemotherapy of Tropical Diseases introduced paromomycin, which was not nationally licensed, and offered it to a number of patients. From 2004 to 2011, 143 case records of amebiasis (123 with amebic colitis, 16 with amebic liver abscess, and 4 with both) in which patients were treated with paromomycin, mainly 1,500 mg/day for 9 or 10 days following metronidazole treatment, were submitted. Among 123 evaluable cases, 23 (18.7%) experienced possible adverse effects, the most common being diarrhea (17/123, 13.8%) and other gastrointestinal problems that were resolved after the completion or discontinuation of treatment. In addition, single cases of bloody stools associated with Clostridium difficile colitis, skin rash, and the elevation of liver enzymes were also reported, although the causal relationship was not clear. HIV infection did not appear to increase the incidence of adverse drug effects. Each of the 11 asymptomatic or mildly symptomatic amebic colitis cases became negative for stool cysts after paromomycin treatment. Paromomycin was shown to be safe and well tolerated, as well as effective in a special subset of amebic colitis cases.

By using the combination of reverse-transcription PCR and rapid amplification of cDNA ends methods, a cDNA encoding mast cell tryptase was successfully cloned from the small intestine of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. The cDNA was 1219 bp long including 810 bp of an open reading frame. Based on the deduced amino acid sequences of known mast cell tryptases of other species, the gerbil mast cell tryptase (gMCT) was highly similar to mouse mast cell protease (mMCP)-7, and seems to be translated as a prepro-enzyme with 25 amino acids of signal and activation peptides and 245 amino acids of mature enzyme. The gMCT mRNA was preferentially transcribed in the intestinal mucosa and to a far lesser extent in the connective tissue such as skin and tongue. Moreover, kinetic study after infection revealed that the amount of gMCT mRNA in the small intestine correlated well with the degree of intestinal mastocytosis. Throughout the course of infection, enzyme-histochemically detectable tryptase activity was limited to mucosal mast cells. Since mucosal mast cells of other rodents, including mice and rats, do not express tryptases, this is the first report of rodent mast cell tryptase expressed in the intestinal mucosa.

Mice lacking functional T and B lymphocytes offer an in vivo animal model for the study of human immune functions. We have attempted to optimize the reconstitution of severe combined immunodeficiency (SCID) mice with human peripheral blood lymphocytes (PBL) using radiation, anti-asialo GM1 antibody or cyclophosphamide (Cy) treatment of the mice and in vitro stimulation of human PBL with interleukin (IL)-2 prior to their transfer to the mice. Total human IgG and tetanus-toxoid (TT)-specific human IgG responses of the mice were used as parameters of successful reconstitution. Treatment of the mice with anti-asialo GM1 antibody significantly enhanced total human IgG levels, but not TT-specific antibody responses, whereas irradiation or Cy treatment of the mice had no effect on human antibody production. In vitro treatment of human PBL with IL-2 prior to engraftment significantly decreased total human IgG responses of human PBL-grafted SCID mice. The immune responses of individual mice within a group were highly variable, which constitutes a major disadvantage of this model.

Malaria remains an important health risk among travelers to tropical/subtropical regions. However, in Japan, only 2 antimalarials are licensed for clinical use - oral quinine and mefloquine. The Research Group on Chemotherapy of Tropical Diseases introduced atovaquone-proguanil in 1999, and reported on its excellent antimalarial efficacy and safety for treating non-immune patients with uncomplicated Plasmodium falciparum malaria (20 adult and 3 pediatric cases) in 2006. In the present study, additional cases of malaria were analyzed to confirm the efficacy and safety of this antimalarial drug. Fourteen adult and 2 pediatric cases of P. falciparum malaria and 13 adult cases and 1 pediatric case of P. vivax/ovale malaria were successfully treated with atovaquone-proguanil, including 3 P. falciparum cases in which the antecedent treatment failed. Two patients with P. vivax malaria were treated twice due to primaquine treatment failure as opposed to atovaquone-proguanil treatment failure. Except for 1 patient with P. falciparum malaria who developed a moderate liver function disturbance, no significant adverse effects were observed. Despite the intrinsic limitations of this study, which was not a formal clinical trial, the data showed that atovaquone-proguanil was an effective and well-tolerated therapeutic option; licensure of this drug in Japan could greatly contribute to individually appropriate treatment options.

Monoclonal anti-idiotypic antibody (Ab2) VF2 was derived from rats immunized with anti-colorectal carcinoma (anti-CRC) monoclonal antibody (Ab1) CO17-1A. In rabbits the Ab2 induced anti anti-idiotypic antibodies (Ab3) that shared idiotopes with the Ab1, bound to the same epitope on CRC cells as Ab1, and bound to the isolated CO17-1A antigen. Monoclonal Ab2 VF2 was superior to the previously described polyclonal goat Ab2 against Ab1 CO17-1A in its capacity to elicit humoral immunity in animals. Ab2 VF2 also induced a specific delayed-type hypersensitivity (DTH) response to challenge with irradiated CO17-1A antigen-positive human CRC cells in mice. Of nine CRC patients immunized with aluminum hydroxide-precipitated Ab2 VF2, six developed antibodies that bound to Ab2, but only three patients developed Ab3 that bound to idiotypic determinants on Ab2. However, the Ab3 did not bind to CO17-1A antigen-positive CRC cells. In contrast, in a previously described trial with polyclonal goat Ab2 to Ab1 CO17-1A, most of the patients developed anti-CRC antibodies. Four of the nine patients immunized with Ab2 VF2 developed DTH responses to intradermal challenge with the Ab2, and in one patient DTH was both Ab2- and antigen-specific. Peripheral blood mononu-clear cells of the four DTH-reactive patients did not proliferate in response to in vitro stimulation with either Ab2 or antigen. These studies demonstrate that the immunomodulatory activity of monoclonal Ab2 VF2 in animals is only in part predictive of its activity in patients.

We report the case of a 62-year-old man who developed eosinophilic pneumonia due to visceral larva migrans (VLM) that was possibly caused by Ascaris suum. The patient, a resident of the middle Kyushu area who was found of eating raw porcine liver, complained of dry cough without dyspnea. The chest radiography showed a migration of infiltrative shadow. Transbronchial lung biopsy of the right middle lobe revealed massive infiltration of eosinophils. The multi-dot enzyme-linked immunosorbent assay (ELISA) and microtiter plate ELISA showed positive results for A. suum; therefore, the patient was diagnosed with VLM caused by A. suum. The patient was administered albendazole (600 mg/day) for 28 days; he recovered successfully with no adverse effects except mild liver dysfunction. Several cases of VLM caused by A. suum have been reported in Japan, with a majority of the cases being reported in Kyushu. Careful history taking of the patient's area of residence and dietary habit is essential for the diagnosis of this parasitic disease with underestimated prevalence.

The present study describes the isolation of Fusarium sporotrichioides from a canine cutaneous ulceration. A 2-year-old male Beagle dog weighing 8.6 kg, with a history of immune-mediated hemolytic anemia (IMHA), had been treated with prednisone for 9 months. Physical examination revealed cutaneous ulceration on the left foreleg. Histopathological examination of skin samples from the ulcerative area revealed many branching hyphae surrounding neutrophils. Since itraconazole (ITZ) is recommended for miscellaneous fungal infections, the dog was treated with ITZ. However, the ulcerative lesions did not improve and after 3 weeks of treatment the dog died due to renal failure. No autopsy was performed. Since the isolate recovered from the biopsy specimen was identifi ed as Fusarium species by morphological characteristics, the animal was diag- nosed as having had an infection caused by this mould. The dog ' s prior prednisone treat- ment may have played a role in establishing the fungal infection. Comparative sequence analyses of the ITS regions of the clinical isolate with those in GenBank showed that it was 100% identical to F. sporotrichioides and less than 96% similar to ITS of other Fusarium species. Based on these fi ndings, F. sporotrichioides was established as the etiologic agent of the canine infection, a situation that has not been previously reported in dogs, as well as humans.