Echinococcus granulosus Protoscolex DM9 Protein Shows High Potential for Serodiagnosis of Alveolar Echinococcosis.

Alveolar echinococcosis (AE) caused by infection with E. multilocularis metacestode, represents one of the most fatal helminthic diseases. AE is principally manifested with infiltrative, proliferating hepatic mass, resembling primary hepatocellular carcinoma. Sometimes metastatic lesions are found in nearby or remote tissue. AE diagnosis largely depends on imaging studies, but atypical findings of imaging features frequently require differential diagnosis from other hepatic lesions. Serological tests may provide further evidence, while obtaining reliable AE materials is not easy. In this study, alternative antigens, specific to AE were identified by analyzing E. granulosus protoscolex proteins. An immunoblot analysis of E. granulosus protoscolex showed that a group of low-molecular-weight proteins in the range from 14 kDa to 16 kDa exhibited a sensitive and specific immune response to AE patient sera. Partial purification and proteomic analysis indicated that this protein group contained myosin, tubulin polymerization promoting protein, fatty-acid binding protein, uncharacterized DM9, heat shock protein 90 cochaperone tebp P-23, and antigen S. When the serological applicability of recombinant forms of these proteins was assessed using enzyme-linked immunosorbent assay, DM9 protein (rEgDM9) showed 90.1% sensitivity (73/81 sera tested) and 94.5% specificity (172/181 sera tested), respectively. rEgDM9 showed weak cross-reactions with patient sera from the transitional and chronic stages of cystic echinococcosis (3 to 5 stages). rEgDM9 would serve as a useful alternative antigen for serodiagnosis of both early- and advanced-stage AE cases.

Comparison of Secretome Profile of Pathogenic and Non-Pathogenic Entamoeba histolytica.

The obligatory intracellular protozoan parasite Entamoeba histolytica causes amebic dysentery and liver abscess. E. histolytica adheres to the host tissues in a contact-dependent manner. E. histolytica excretory-secretory products (ESP) might play critical roles during invasion. We comparatively analyzed the secretome profile of E. histolytica pathogenic HM-1:IMSS and non-pathogenic Rahman strains. The two ESP revealed similar but distinct spotting patterns. In both ESP, alcohol dehydrogenase, enolase 1, and transketolase, which control classical carbohydrate metabolism and other moonlighting effects, constituted the most abundant fractions. We recognized differently secreted molecules. Secretion of cytoskeletal organization proteins (actin, actin binding protein, and EHI_068510), protein remodeling amino peptidase, and multifunctional elongation factor 1-α were increased in Rahman. Conversely, carbohydrate metabolizing enolase 1, alcohol dehydrogenase, transketolase, calponin, phosphoglucose mutase, malic enzyme and EHI_156420, xenobiotic scavenging superoxide dismutase and EHI_140740, and pyruvate:ferredoxin oxidoreductase and coronin (carbohydrate metabolism/detoxification) showed reduced secretion. Transcription levels of some genes involved in these processes also decreased. Changes of secretory behavior, especially decreased secretion of multifunctional carbohydrate metabolizing enzymes and detoxifying proteins that importantly participated in amoeba pathogenesis might reflect avirulent nature of Rahman strain in the host.

Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure.

Rec8 is a prominent component of the meiotic prophase chromosome axis that mediates sister chromatid cohesion, homologous recombination and chromosome synapsis. Here, we explore the prophase roles of Rec8. (i) During the meiotic divisions, Rec8 phosphorylation mediates its separase-mediated cleavage. We show here that such cleavage plays no detectable role for chromosomal events of prophase. (ii) We have analyzed in detail three rec8 phospho-mutants, with 6, 24 or 29 alanine substitutions. A distinct 'separation of function' phenotype is revealed. In the mutants, axis formation and recombination initiation are normal, as is non-crossover recombination; in contrast, crossover (CO)-related events are defective. Moreover, the severities of these defects increase coordinately with the number of substitution mutations, consistent with the possibility that global phosphorylation of Rec8 is important for these effects. (iii) We have analyzed the roles of three kinases that phosphorylate Rec8 during prophase. Timed inhibition of Dbf4-dependent Cdc7 kinase confers defects concordant with rec8 phospho-mutant phenotypes. Inhibition of Hrr25 or Cdc5/polo-like kinase does not. Our results suggest that Rec8's prophase function, independently of cohesin cleavage, contributes to CO-specific events in conjunction with the maintenance of homolog bias at the leptotene/zygotene transition of meiotic prophase.

Human Sparganosis in Korea.

BACKGROUND: Sparganosis is a larval cestodiasis caused by the plerocercoid of Spirometra spp. Since the first description of human sparganosis in 1924, several hundred cases have been reported in Korea. However, systematic approaches for literature surveys of Korean sparganosis have seldom appeared. METHODS: We searched publicly available databases such as PubMed, Research Information Sharing Service, and Korea Medical Citation Index with relevant Medical Subject Headings. RESULTS: At least 438 Korean sparganosis cases have been described from 1924 to 2015. Preoperative diagnosis has been significantly increased since the 1980s due to popularization of serological and imaging diagnostics. Cases were largely detected from fifth decades in general, but cerebral sparganosis was detected in relatively young age groups (third and fourth decades). Sparganosis was prevalent in men (75.9%). Consumption of frog/snake and drinking unfiltered water were found in 63.4% and 16.9% of patients, respectively. Most frequently affected sites were subcutaneous tissues (49.9%), followed by the central nervous system (36.2%). Involvements of visceral organs (7.6%), ocular regions (3.6%), and muscles (2.7%) were noticed. In women, breast sparganosis constituted a large proportion (34.2%). Sparganosis associated with immunocompromised patients has recently been reported. CONCLUSION: Sparganosis has been continuously reported in Korea during the past 90 years, although its incidence has decreased during the last 20 years. The disease is mostly characterized by subcutaneous nodule, but infection of the worm in vital organs often results in serious illness. Continuous awareness is warranted to monitor sparganosis occurrence and associated clinical consequences.

Biochemical Characterization of Echinococcus multilocularis Antigen B3 Reveals Insight into Adaptation and Maintenance of Parasitic Homeostasis at the Host-Parasite Interface.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis metacestode is frequently associated with deleterious zoonotic helminthiasis. The growth patterns and morphological features of AE, such as invasion of the liver parenchyme and multiplication into multivesiculated masses, are similar to those of malignant tumors. AE has been increasingly detected in several regions of Europe, North America, Central Asia, and northwestern China. An isoform of E. multilocularis antigen B3 (EmAgB3) shows a specific immunoreactivity against patient sera of active-stage AE, suggesting that EmAgB3 might play important roles during adaptation of the parasite to hosts. However, expression patterns and biochemical properties of EmAgB3 remained elusive. The protein profile and nature of component proteins of E. multilocularis hydatid fluid (EmHF) have never been addressed. In this study, we conducted proteome analysis of EmHF of AE cysts harvested from immunocompetent mice. We observed the molecular and biochemical properties of EmAgB3, including differential transcription patterns of paralogous genes, macromolecular protein status by self-assembly, distinct oligomeric states according to individual anatomical compartments of the worm, and hydrophobic ligand-binding protein activity. We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. EmAgB3 might participate in immune response and recruitment of essential host lipids at the host-parasite interface. Our results might contribute to an in depth understanding of the biophysical and biological features of EmAgB3, thus providing insights into the design of novel targets to control AE.

Spatiotemporal Expression Patterns and Antibody Reactivity of Taeniidae Endophilin B1.

Larval Taeniidae, such as metacestodes of Taenia solium, Echinococcus granulosus, and Echinococcus multilocularis, produce chronic and fatal helminthic diseases. Proper identification of these zoonotic cestodiases is often challenging and is hampered in some clinical settings. Endophilin B1 plays critical roles in the maintenance of membrane contours and endocytosis. We isolated proteins homologous to endophilin B1 from T. solium, Taenia saginata, and Taenia asiatica The three Taeniidae endophilin B1 proteins shared 92.9 to 96.6% sequence identity. They harbored a Bin1/amphiphysin/Rvs (BAR) domain and residues for a dimeric interface but lacked a SRC homology 3 (SH3) domain. Endophilin B1 showed a unique immunological profile and was abundantly expressed in the tegumental syncytium of Taeniidae metacestodes and adults. Bacterially expressed recombinant T. solium endophilin B1 (rTsMEndoB1) demonstrated a sensitivity of 79.7% (345/433 cases) for serodiagnosis of larval Taeniidae infections. The protein showed strong immune recognition patterns against sera from patients with chronic neurocysticercosis, cystic echinococcosis, or advanced-stage alveolar echinococcosis. Adult Taeniidae infections exhibited moderate degrees of positive antibody responses (65.7% [23/35 samples]). rTsMEndoB1 showed some cross-reactivity with sera from patients infected with Diphyllobothriidae (23.6% [25/106 samples]) but not with sera from patients with other parasitic diseases or normal controls. The specificity was 91.7% (256/301 samples). The positive and negative predictive values were 93.6% and 73.4%, respectively. Our results demonstrate that Taeniidae endophilin B1 may be involved in the control of membrane dynamics, thus contributing to shaping and maintaining the tegumental curvature. rTsMEndoB1 may be useful for large-scale screening, as well as for individual diagnosis and follow-up surveillance of Taeniidae infections.

An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis.

Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.

Egg-specific expression of protein with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis.

Despite recent reports regarding the biology of cytosine methylation in Schistosoma mansoni, the impact of the regulatory machinery remains unclear in diverse platyhelminthes. This ambiguity is reinforced by discoveries of DNA methyltransferase 2 (DNMT2)-only organisms and the substrate specificity of DNMT2 preferential to RNA molecules. Here, we characterized a novel DNA methyltransferase, named CsDNMT2, in a liver fluke Clonorchis sinensis. The protein exhibited structural properties conserved in other members of the DNMT2 family. The native and recombinant CsDNMT2 exhibited considerable enzymatic activity on DNA. The spatiotemporal expression of CsDNMT2 mirrored that of 5-methylcytosine (5 mC), both of which were elevated in the C. sinensis eggs. However, CsDNMT2 and 5 mC were marginally detected in other histological regions of C. sinensis adults including ovaries and seminal receptacle. The methylation site seemed not related to genomic loci occupied by progenies of an active long-terminal-repeat retrotransposon. Taken together, our data strongly suggest that C. sinensis has preserved the functional DNA methylation machinery and that DNMT2 acts as a genuine alternative to DNMT1/DNMT3 to methylate DNA in the DNMT2-only organism. The epigenetic regulation would target functional genes primarily involved in the formation and/or maturation of eggs, rather than retrotransposons.

Taenia solium metacestode fasciclin-like protein is reactive with sera of chronic neurocysticercosis.

OBJECTIVES: Neurocysticercosis (NC), an infection of the central nervous system with Taenia solium metacestodes (TsM), invokes a formidable neurological disease. A bundle of antigens is applicable for serodiagnosis of active cases, while they demonstrate fairly low reactivity against sera of chronic NC. Identification of sensitive biomarkers for chronic NC is critical for appropriate management of patients. METHODS: Proteome analysis revealed several isoforms of 65- and 83-kDa TsM fasciclin-like proteins (TsMFas) to be highly reactive with sera of chronic NC. A cDNA encoding one of the 83-kDa TsMFas (TsMFas1) was isolated from a cDNA library. We expressed a recombinant protein (rTsMFas1) and evaluated its diagnostic potential employing sera from chronic NC (n = 80), tissue-invasive cestodiases (n = 169) and trematodiases (n = 80) and those of normal controls (n = 50). RESULTS: Secretory TsMFas1 was composed of 766 amino acid polypeptide and harboured fasciclin and fasciclin-superfamily domains. The protein was constitutively expressed in metacestode and adult stages, with preferential locality in the scolex. Bacterially expressed rTsMFas1 exhibited 78.8% sensitivity (63/80 cases) and 93% specificity (278/299 samples) in diagnosing chronic NC. Some cross-reactivity was observed with sera of cystic echinococcosis (10/56, 17.8%) and sparganosis (4/50, 8%). Positive and negative predictive values were 75% and 95.5%, respectively. CONCLUSION: TsM fasciclin-like protein may be useful for differential diagnosis of chronic NC in clinical settings, especially where both NC and other infectious cerebral granulomatoses are prevalent.

TSH receptor antibody as a predictor of difficult robotic thyroidectomy in patients with Graves' disease.

Thyroidectomy in Graves' disease can be challenging due to greater thyroid size and vascularity. While thyroid stimulating hormone receptor antibody (TRAb) level is associated with disease severity and thyroid vascularity, its impact on operative outcomes remains unclear. This study aimed to compare challenging factors for robotic thyroidectomy (RT) and open thyroidectomy (OT) in Graves' disease patients, including TRAb as a predictive factor for difficult thyroidectomy. This retrospective study included Graves' disease patients who underwent total thyroidectomy between September 2013 and January 2023. The clinical characteristics and operative outcomes were compared between patients who received OT and bilateral axillo-breast approach RT. Factors affecting operation time and estimated blood loss (EBL) were evaluated in both groups using regression analyses. A total of 85 patients received either OT (n = 48) or RT (n = 37). Median thyroid volumes in the OT and RT groups were 72.4 g and 57.6 g, respectively. Operation time was affected by thyroid volume in both groups. Additionally, higher thyroid hormone levels and bilateral central neck node dissection prolonged operation time in the RT group. EBL was marginally associated with thyroid volume in the OT group. However, in the RT group, TRAb level was independently associated with greater EBL (p = 0.04), while no significant association was found with thyroid volume. Predictive factors for difficult thyroidectomy differed by operation approaches. TRAb significantly predicted intraoperative bleeding in RT, while this association was absent in OT. Caution is warranted when performing RT on Graves' disease patients with high TRAb levels.

Outcomes of bilateral axillo-breast approach robotic parathyroidectomy versus open parathyroidectomy for primary hyperparathyroidism: a single-institution retrospective study.

Purpose: Bilateral axillo-breast approach robotic parathyroidectomy (BABA-RP) aims to remove overactive or enlarged parathyroid glands with no visible neck collar incision. In this study, we compared the safety and surgical outcomes of BABA-RP vs. those of an open surgery group to ascertain whether BABA-RP is a safe and feasible surgical approach for patients with primary hyperparathyroidism (pHPT). Methods: This single-institution retrospective cohort study included 74 patients with primary HPT who underwent open parathyroidectomy (n = 37) or BABA-RP (n = 37) at our institution between November 2014 and March 2023. Patient demographics, biochemical cure rates, operative time, blood loss rates, and complication rates were examined and compared. Results: The patients in the BABA-RP group were younger and had a longer mean operative time. Regarding complication events, 2 patients in the open surgery group and 1 patient in the BABA-RP group had transient hypoparathyroidism. All 74 patients achieved biochemical cure at <6 months, regardless of the approach used. Two patients in the BABA-RP group and 1 patient in the open surgery group had carcinoma on surgical pathology. All 3 patients with parathyroid carcinoma remained recurrence-free at 1-year follow-up. Conclusion: Compared with the open procedure, BABA-RP is a safe and feasible procedure that provides an excellent biochemical cure rate for patients with pHPT and has superior cosmetic benefits with equivalent surgical outcomes.

Evaluation of IgG4 subclass antibody detection by peptide-based ELISA for the diagnosis of human paragonimiasis heterotrema.

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.

Tetrathyridia of Mesocestoides lineatus in Chinese snakes and their adults recovered from experimental animals.

Morphological characteristics of Mesocestoides lineatus tetrathyridia collected from Chinese snakes and their adults recovered from experimental animals were studied. The tetrathyridia were detected mainly in the mesentery of 2 snake species, Agkistrodon saxatilis (25%) and Elaphe schrenckii (20%). They were 1.73 by 1.02 mm in average size and had an invaginated scolex with 4 suckers. Adult tapeworms were recovered from 2 hamsters and 1 dog, which were orally infected with 5-10 larvae each. Adults from hamsters were about 32 cm long and those from a dog were about 58 cm long. The scolex was 0.56 mm in average width with 4 suckers of 0.17 by 0.15 mm in average size. Mature proglottids measured 0.29 by 0.91 mm (av.). Ovaries and vitellaria bilobed and located in the posterior portion of proglottids. The cirrus sac was oval-shaped and located median. Testes were follicular, distributed in both lateral fields of proglottids, and 41-52 in number per proglottid. Gravid proglottids were 1.84 by 1.39 mm (av.) with a characteristic paruterine organ. Eggs were 35 by 27 µm in average size with a hexacanth embryo. These morphological characteristics of adult worms were identical with those of M. lineatus reported previously. Therefore, it has been confirmed that the tetrathyridia detected in 2 species of Chinese snakes are the metacestodes of M. lineatus, and 2 snake species, A. saxatilis and E. schrenckii, play the role of intermediate hosts.

Omega-Class Glutathione Transferases Protect DNA from Oxidative Stress in Pathogenic Helminth Reproductive Cells.

Pathogenic helminths have evolved mechanisms to preserve reproductive function while surviving long-term in the host via robust protective responses. A protective role of antioxidant enzymes in preventing DNA degradation has long been proposed, but little evidence has been provided. Here, we show that omega-class glutathione transferases (GSTOs) are critical for maintaining viability by protecting the reproductive cell DNA of the carcinogenic liver fluke, Clonorchis sinensis. Clonorchis sinensis GSTO (CsGSTO) activities modified by changes in the GSH/GSSG and NADPH/NADP+ molar ratios suppressed the overproduction of reactive oxygen species. CsGSTO1 and CsGSTO2 catalyzed deglutathionylation under physiologic and low-stress conditions (GSH/GSSG ratio of 6:1 or higher) but promoted glutathionylation under high-stress conditions (GSH/GSSG ratio of 3:1 or lower). Gliotoxin-induced functional disruption of CsGSTOs in living C. sinensis reduced the GSH/GSSG molar ratio and increased the production of protein glutathionylation (PSSG) under physiologic and low-stress conditions, indicating that suppression of GSTO function did not affect deglutathionylation. However, the perturbation of CsGSTOs decreased the GSH/GSSG ratio but also reduced PSSG production under high oxidative stress, demonstrating that glutathionylation was impeded. In response to oxidative stimuli, C. sinensis decreased GSTO-specific dehydroascorbate reductase and thiol transferase activities and the GSH/GSSG ratio, while it increased the NADPH/NADP+ ratio and PSSG. CsGSTOs utilized GSH to regulate GSH/GSSG and NADPH/NADP+ recycling and triggered a redox signal leading to nuclear translocation. Nuclear-imported CsGSTOs were modified by glutathionylation to prevent DNA damage. Antibodies specific to CsGSTOs dose-dependently inhibited this process. Disruption of CsGSTOs or the depletion of GSH caused glutathionylation defects, leading to DNA degradation. Our results demonstrate that CsGSTOs and the GSH system play a previously unappreciated role in protecting DNA from oxidative stress.

Experimental murine fascioliasis derives early immune suppression with increased levels of TGF-ß and IL-4.

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19(+) B cells was observed as early as week 1 post-infection while CD4(+)/CD8(+) T cells were down-regulated. Accumulation of Mac1(+) cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1ß expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-ß were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-ß and IL-4 during the early stages of infection.

Cerebral paragonimiasis: Clinicoradiological features and serodiagnosis using recombinant yolk ferritin.

Cerebral paragonimiasis (CP), caused by aberrant migration of Paragonimus worms, frequently invokes serious illness. The causal relationship between the lesion characteristics and patients' symptoms has poorly been understood. CP serodiagnosis has not been properly evaluated to date. A total of 111 CP cases were diagnosed in our laboratory between 1982 and 2003. This study retrospectively assessed the clinical and imaging characteristics of the 105 patients along with the evaluation of diagnostic potentials of recombinant P. westermani yolk ferritin (rPwYF) by enzyme-linked immunosorbent assay (ELISA) employing patients' sera and cerebrospinal fluids (CSFs). We analyzed 60 male and 45 female patients; 50 early-stage patients with non-calcified enhancing nodule(s) (median age, 38 years; interquartile range [IQR], 24.75-52; median symptom duration, 0.75 years; IQR, 0.2-2) and 54 chronic cases having calcified lesion(s) (median age, 33 years; IQR, 25-41; median symptom duration, 10 years; IQR, 5-20). One patient showed a normal neuroimage. The patients were largely diagnosed in their 30s. The parietal lobe was most commonly affected, followed by occipital, frontal, and temporal lobes. Twenty-six patients had lesions encompassing ≥ two lobes. The patients complained mainly of seizures, headaches, hemiparesis, and focal neurologic deficits (P < 0.001). Seizures and visual defects were predominant in patients with calcified lesion(s) (P < 0.001). The diagnostic sensitivity and specificity of rPwYF against serum/CSF were 100%/97% and 97.2%/92.5%, respectively. The specific IgG antibody levels against rPwYF in sera and CSFs showed a positive correlation (r = 0.59). The clinical manifestations of the early-stage patients might be associated with cortical lesions or meningeal irritation, while those in the chronic stage were caused by conglomerated space-occupying lesions. rPwYF would be useful for the serodiagnosis of both early and chronic CP cases.

Comparison of the antibody responses to Plasmodium vivax and Plasmodium falciparum antigens in residents of Mandalay, Myanmar.

BACKGROUND: The aim of this study was to investigate the profile of antibodies against several antigens of Plasmodium vivax and Plasmodium falciparum in Mandalay, Myanmar. METHODS: Malaria parasites were identified by microscopic examination. To test the antibodies against P. vivax and P. falciparum in sera, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood parasite antigens. An enzyme-linked immunosorbent assay (ELISA) was performed with circumsporozoite protein (CSP), Pvs25 and Pvs28 recombinant proteins of transmission-blocking vaccine candidates for P. vivax, and liver stage specific antigen-1 and -3 (PfLSA-1, PfLSA-3) for P. falciparum. RESULTS: Fourteen patients among 112 were found to be infected with P. vivax and 26 with P. falciparum by thick smear examination. Twenty-three patients were found to be infected with P. vivax, 19 with P. falciparum and five with both by thin smear examination. Blood samples were divided into two groups: Group I consisted of patients who were positive for infection by microscopic examination, and Group II consisted of those who showed symptoms, but were negative in microscopic examination. In P. falciparum, IgG against the blood stage antigen in Group I (80.8%) was higher than in Group II (70.0%). In P. vivax, IgG against the blood stage antigen in Group I (53.8%) was higher than in Group II (41.7%). However, the positivity rate of the PvCSP VK210 subtype in Group II (40.0%) was higher than in Group I (23.1%). Similarly for the PvCSP VK247 subtype, Group II (21.7%) was higher than that for Group I (9.6%). A similar pattern was observed in the ELISA using Pvs25 and Pvs28: positive rates of Group II were higher than those for Group I. However, those differences were not shown significant in statistics. CONCLUSIONS: The positive rates for blood stage antigens of P. falciparum were higher in Group I than in Group II, but the positive rates for antigens of other stages (PfLSA-1 and -3) showed opposite results. Similar to P. falciparum, the positive rate of pre-blood stage (CSP VK210 and 247 subtype) and post-blood stage (Pvs25 and 28) antigens of P. vivax were higher in Group II than in Group I. Therefore, sero-diagnosis is not helpful to discriminate between malaria patients and symptomatic individuals during the epidemic season in Myanmar.

Plasmodium vivax: collaborative roles for plasmepsin 4 and vivapains in hemoglobin hydrolysis.

Plasmepsins, a family of aspartic proteases of Plasmodium species, are known to participate in a wide variety of cellular processes essential for parasite survival. Therefore, the plasmepsins of malaria parasites have been recognized as attractive antimalarial drug targets. Although the plasmepsins of P. falciparum have been extensively characterized, the plasmepsins of P. vivax are currently not well known. To expand our understanding of the plasmepsins of P. vivax, we characterized plasmepsin 4 of P. vivax (PvPM4). The bacterially expressed recombinant PvPM4 was insoluble, but it was easily refolded into a soluble protein. The processing of PvPM4 into a mature enzyme occurred through autocatalytic activity under acidic conditions in a pepstatin A-sensitive manner, in which process a portion of prodomain was essential for correct folding. PvPM4 could hydrolyze native human hemoglobin at acidic pHs, but preferred denatured hemoglobin as a substrate. PvPM4 acted synergistically with vivapain-2 and vivapain-3, cysteine proteases of P. vivax, in the hydrolysis of hemoglobin. The vivapains also mediated processing of PvPM4 into a mature enzyme. These results collectively suggest that PvPM4 is an active hemoglobinase of P. vivax that works collaboratively with vivapains to enhance the parasite's ability to hydrolyze hemoglobin.

Prevalence of Clonorchis sinensis metacercariae in freshwater fish from three latitudinal regions of the Korean Peninsula.

A large-scale survey was conducted to investigate the infection status of fresh water fishes with Clonorchis sinensis metacercariae (CsMc) in 3 wide regions, which were tentatively divided by latitudinal levels of the Korean peninsula. A total of 4,071 freshwater fishes were collected from 3 regions, i.e., northern (Gangwon-do: 1,543 fish), middle (Chungcheongbuk-do and Gyeongsangbuk-do: 1,167 fish), and southern areas (Jeollanam-do, Ulsan-si, and Gyeongsangnam-do: 1,361 fish). Each fish was examined by the artificial digestion method from 2003 to 2010. In northern areas, only 11 (0.7%) fish of 2 species, Pungtungia herzi and Squalidus japonicus coreanus from Hantan-gang, Cheolwon-gun, Gangwon-do were infected with av. 2.6 CsMc. In middle areas, 149 (12.8%) fish were infected with av. 164 CsMc. In southern areas, 538 (39.5%) fish were infected with av. 159 CsMc. In the analysis of endemicity in 3 regions with an index fish, P. herzi, 9 (6.2%) of 146 P. herzi from northern areas were infected with av. 2.8 CsMc. In middle areas, 34 (31.8%) of 107 P. herzi were infected with av. 215 CsMc, and in southern areas, 158 (92.9%) of 170 P. herzi were infected with av. 409 CsMc. From these results, it has been confirmed that the infection status of fish with CsMc is obviously different among the 3 latitudinal regions of the Korean peninsula with higher prevalence and burden in southern regions.