Clonorchis sinensis omega-class glutathione transferases are reliable biomarkers for serodiagnosis of clonorchiasis and opisthorchiasis.

OBJECTIVES: To determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections. METHODS: Specific antibody levels against rCsGSTo1 and rCsGSTo2 in patients' sera of egg-positive opisthorchiasis (n = 87) and clonorchiasis (n = 120), as well as those in sera from patients with other helminthic infections (n = 252) and healthy controls (n = 40) were retrospectively analysed by ELISA. RESULTS: We observed highly positive correlation coefficients between specific antibody levels against rCsGSTo1 and rCsGSTo2 and egg counts per gramme of faeces (EPG) of patients with opisthorchiasis (n = 87; r = 0.88 for rCsGSTo1 and r = 0.90 for rCsGSTo2). Sera from opisthorchiasis patients whose EPG counts >100 (n = 43) revealed high antibody titres against both antigens. Patients' sera with low EPG counts (<100, n = 44) also exhibited reliable sensitivities of 93.2% and 97.7% for rCsGSTo1 and rCsGSTo2, respectively. Sera from clonorchiasis patients showed sensitivities of 90% (108/120 samples) and 89.2% (107/120 sera) for rCsGSTo1 and rCsGSTo2. Overall diagnostic sensitivities for liver-fluke infections were 92.3% for rCsGSTo1 (191/207 samples) and 93.2% for rCsGSTo2 (193/207 samples). Specificities were 89.7% (rCsGSTo1) and 97.6% (rCsGSTo2). CONCLUSIONS: Detection of specific antibody levels against rCsGSTo1 or rCsGSTo2 might be promising for the serodiagnosis of patients infected with these two phylogenetically close carcinogenic liver-flukes.

Fish-borne nematodiases in South America: neglected emerging diseases.

Fish-borne zoonotic nematodes may infect humans when fish or squid are ingested raw or inadequately cooked. Human infections may have serious consequences, including the unexpected deaths of infected people. This kind of disease is poorly known in general, and the characteristics of such infections in South American countries as a whole have never been assessed. In this paper the present status of fish-borne nematodiases in humans in South American countries is characterized. Potentially zoonotic nematode species are very common in both freshwater and marine fish in South America. Reports of human infections have only been found in some countries, and their incidence (especially with anisakids and Gnathostoma spp.) varies from country to country. Apparently they are more abundant in countries with strong traditions of eating raw fish, and are more frequent on the western coast of South America. So far fish-borne nematodes have been reported in Argentina, Brazil, Chile, Colombia, Ecuador and Peru. In recent years, cases of human infection have appeared in probably underestimated numbers. People need to be clearly informed about risky feeding habits, and physicians need to learn more about zoonotic diseases.

Molecular identification of the trematode Paragonimus in faecal samples from the wild cat Prionailurus bengalensis in the Da Krong Nature Reserve, Vietnam.

Conventional identification of Paragonimus species and their natural definitive hosts is based on the morphological features of adult parasites isolated from the lungs of wild mammalian hosts. However, wild animals are protected by strict regulations and sampling is not always possible. Recently, molecular techniques have been developed to identify the internal transcribed spacer (ITS) sequences of Paragonimus eggs in faeces/sputum of human patients. Also, mammalian hosts can be identified using the D-loop sequence of mitochondrial DNA in faecal samples. In this study, we used molecular techniques on faeces from wild animals collected in Da Krong Nature Reserve, Quang Tri province, central Vietnam, where Paragonimus metacercariae are highly prevalent in mountain crabs, to identify Paragonimus species and their natural definitive hosts. The results indicated that wild cats, Prionailurus bengalensis, were infected with at least three different Paragonimus species, P. westermani, P. skrjabini and P. heterotremus. Because all of these species can infect humans in Asian countries, human paragonimiasis should be considered in this area.

Sinusoidal obstruction syndrome after allogeneic hematopoietic stem cell transplantation: Incidence, risk factors and outcomes.

This retrospective study was conducted in Japan to determine the incidence, risk factors and outcomes of sinusoidal obstruction syndrome (SOS) after allogeneic hematopoietic stem cell transplantation (HSCT). Among 4290 patients undergoing allogeneic HSCT between 1999 and 2010, 462 were diagnosed with SOS according to the Seattle criteria (cumulative incidence, 10.8%). The cumulative incidence of SOS diagnosed by the modified Seattle criteria was 9.3%. Of 462 patients, 107 met the Baltimore criteria and 168 had severe SOS with renal and/or respiratory failure. The median onset for SOS was 12 days after HSCT (range, -2-30). Overall survival at day 100 was 32% for SOS and 15% for severe SOS. Multivariate analyses showed that significant independent risk factors for SOS were the number of HSCTs, age, performance status, hepatitis C virus-seropositivity, advanced disease status and myeloablative regimen. SOS was highly associated with overall mortality (hazard ratio, 2.09; P<0.001). Our retrospective survey showed that the cumulative incidence of SOS in Japan was 10.8%, similar to that previously reported in Western countries, and that the overall survival of patients who developed SOS was low. Furthermore, several risk factors were identified. Preventive and therapeutic strategies for high-risk SOS patients must be established to improve overall survival.

Patient-reported quality of life after allogeneic hematopoietic cell transplantation or chemotherapy for acute leukemia.

When discussing treatment options for patients with acute leukemia, it is important to acknowledge the impact of allogeneic hematopoietic cell transplantation (allo-HCT) or chemotherapy on quality of life (QOL). We performed a cross-sectional questionnaire study that administered SF-36, FACT-Leukemia and EuroQOL5D to 524 acute leukemia survivors, to compare patient-reported QOL between chemotherapy and allo-HCT, and to elucidate predictors of QOL. Patients who received chemotherapy alone had a better physical QOL than those who received allo-HCT. On the other hand, the allo-HCT group reported a better mental QOL. In the comparison of QOL in the allo-HCT patients according to the presence of GVHD at survey, patients who had GVHD symptoms experienced statistically and clinically significantly worse QOL than those who did not. In the allo-HCT patients without GVHD, the physical QOL was comparable to that in the chemotherapy patients, and they experienced significantly better mental and general QOL than the chemotherapy patients. GVHD and immunosuppressive drugs at survey were strongly associated with worse QOL after allo-HCT. In the chemotherapy group, a shorter time between treatment completion and survey was significantly associated with worse QOL. Further evaluation of QOL by a longitudinal assessment with quantitative and qualitative analyses are warranted.

Is Opisthorchis viverrini an avian liver fluke?

Recently, in the Journal of Helminthology (May 2013), Dao et al. reported that Opisthorchis viverrini-like flukes were found in the bile duct of domestic ducks in Vietnam. They stated that this is the first record of Opisthorchis sp. in birds in Vietnam. However, three Opisthorchis species--O. cheelis, O. longissimus and O. parageminus--in birds in Vietnam were described by Le in 2000. Amongst these, O. parageminus was first reported, by Oshmarin in 1970, as a new Opisthorchis species found in domestic ducks (Anas platyrhynchos) in Vietnam. Morphologially O. viverrini-like flukes described by Dao et al. are much more similar to O. parageminus than to O. viverrini. The phylogenetic trees of internal transcribed spacer 2 (ITS2) and cytochrome oxidase 1 (CO1) gene sequences also showed that the O. viverrini-like liver flukes from domestic ducks were closer to O. lobatus than to O. viverrini. Therefore, O. viverrini-like liver flukes reported by Dao et al. (2013) are most likely to be O. parageminus.

Metacercarial polymorphism and genetic variation of Paragonimus heterotremus (Digenea: Paragonimidae), and a re-appraisal of the taxonomic status of Paragonimus pseudoheterotremus.

Paragonimus heterotremus, which is an important pathogen for human paragonimiasis in Asia, is recognized as having the smallest metacercariae (maximum diameter < 300 µm) of any previously reported Paragonimus species. Recently, P. pseudoheterotremus has been described from Thailand as a new species having metacercariae (about 200 µm) slightly smaller than those of Thai P. heterotremus. In fact, the small size of P. pseudoheterotremus metacercariae is compatible with those of P. heterotremus from India and China. In this study in Vietnam, we found variably sized small metacercariae which are expected to consist of both P. heterotremus and P. pseudoheterotremus. Contrary to expectation, the adult flukes obtained by separate infection of experimental cats with different sized metacercariae were all identified as P. heterotremus, using both morphological and molecular characteristics. The molecular analyses of an extensive collection of P. heterotremus/P. pseudoheterotremus isolates from Asian countries also indicated that genetic distances between different populations of P. heterotremus are even larger than that between P. pseudoheterotremus and P. heterotremus. The haplotype network showed that all P. heterotremus and P. pseudoheterotremus isolates formed a P. heterotremus complex consisting of three groups with strong geographical origins. In addition, the Indian P. heterotremus group is the root of the other P. heterotremus and P. pseudoheterotremus populations. Based on the observed metacercarial polymorphisms and genetic variation in P. heterotremus, P. pseudoheterotremus should be considered a geographically isolated population of the P. heterotremus complex.

Effect of related donor availability on outcome of AML in the context of related and unrelated hematopoietic cell transplantation.

Although allogeneic hematopoietic cell transplantation (HCT) from a related donor is effective therapy for younger patients with AML, it remains unknown how the availability of a related donor affects the outcome when unrelated HCT is a treatment option for patients without a related donor. To address this issue, we retrospectively analyzed 605 cytogenetically non-favorable AML patients younger than 50 years for whom a related donor search was performed during first CR (CR1). The 4-year OS was 62% in 253 patients with a related donor and 59% in 352 patients without a related donor (P=0.534). Allogeneic HCT was performed during CR1 in 62% and 41% of patients with and without a related donor, respectively. Among patients transplanted in CR1, the cumulative incidence of non-relapse mortality was significantly higher in patients without a related donor (P=0.022), but there was no difference in post-transplant OS between the groups (P=0.262). These findings show the usefulness of unrelated HCT in younger patients with cytogenetically non-favorable AML who do not have a related donor. The extensive use of unrelated HCT for such patients may minimize the potential disadvantage of lacking a related donor.

Genetically variant populations of Paragonimus proliferus Hsia & Chen, 1964 from central Vietnam.

Among about 50 nominal Paragonimus species, Paragonimus proliferus is rather a rare species, found only in Yunnan province, China, until our recent discovery of this species in Lai Chau province, northern Vietnam close to Yunnan, China. Here we add Quang Binh province, central Vietnam as a new endemic area of P. proliferus. Large excysted metacercariae found in mountainous crabs, Potamiscus tannanti, were morphologically identified as P. proliferus, which was confirmed further by molecular analyses. Second internal transcribed spacer (ITS2) sequences of the P. proliferus population in Quang Binh province were completely (100%) identical with those of P. proliferus populations in Lai Chau province, northern Vietnam and Yunnan province, China. However, cytochrome oxidase subunit 1 (CO1) gene sequences of Quang Binh population were significantly different (5.6%) from that of previously reported northern Vietnam and Yunnan, China populations. A phylogenetic tree revealed that all CO1 sequences of P. proliferus Quang Binh population formed a distinct group, which was clustered with northern Vietnam and Yunnan, China populations with the bootstrap value of 75%. This is the first record of the genetically variant population of P. proliferus, distribution of which is geographically remote from the previously reported endemic areas in the border between northern Vietnam and Yunnan, China, suggesting that P. proliferus may be much more widely distributed in the Indochina peninsula (or South-East Asia) than expected.

Discovery of Paragonimus skrjabini in Vietnam and its phylogenetic status in the Paragonimus skrjabini complex.

Two members of the Paragonimus skrjabini complex, P. skrjabini and P. miyazakii, are now considered as two sub-species, P. skrjabini skrjabini and P. skrjabini miyazakii. They are well known as important pathogens for human paragonimiasis in China and Japan. Recently, members of this species complex have been reported from India. Here we report the first discovery of P. skrjabini from freshwater crab hosts in Thanh Hoa province, Vietnam. For morphological and molecular phylogenetic studies, adult worms were obtained by experimental infection in cats and dogs. Molecular analyses of metacercariae and adults revealed that the P. skrjabini population from Thanh Hoa, Vietnam was almost completely identical with that from Yunnan province, China. Those populations from Thanh Hoa, Vietnam and Yunnan, China and those from Manipur, India were significantly different from P. skrjabini populations reported from other localities of China in cytochrome oxidase subunit 1 (CO1) gene sequences, indicating considerable genetic variation within the P. skrjabini complex. Moreover, low bootstrap values in the CO1 tree suggested that more variant genotypes belonging to P. skrjabini complex may be found in other Asian countries in between Vietnam and India, such as Myanmar, Laos and Thailand. Since P. skrjabini is known as a pathogen for humans, paragonimiasis cases caused by P. skrjabini might be found in Vietnam and other Asian countries.

Serodiagnostic reliability of single-step enriched low-molecular weight proteins of Taenia solium metacestode of American and Asian isolates.

The low-molecular weight proteins (LMWPs) of Taenia solium metacestode (TsM) constituted pertinent serodiagnostic antigens for cysticercosis. We established a novel single-step purification of the LMWPs from TsM cyst fluid (CF). When the CF was precipitated with trichloroacetic acid/acetone mixture at the final concentrations of 5 and 50%, most LMWPs (ranging 7-38kDa) remained in the supernatant fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses revealed that the LMWPs consisted mainly with the previously reported subunit proteins of the 120- and 150-kDa antigen complexes. Serum samples from neurocysticercosis (NC) and other helminthic infections, as well as those from healthy controls were tested by both immunoblotting and micro-ELISA. In 135 cases of active stage NC patients, 132 cases (97.7%) showed positive reactions. Serum samples from other helminthic diseases (n=125) and healthy controls (n=100) exhibited no positive reactions except for cystic echinococcosis, of which 12% (3/25 cases) exhibited low levels of cross-reactivity. The LMWPs from different geographical regions (Korea and Mexico) showed diagnostic sensitivity and specificity of 97.7% and 98.7% against active stage NC. Our single-step separation method for the LMWPs provided excellent performance with easy applicability and high reproducibility, which has a great benefit for preparation of potent antigen in endemic areas.

1,5-Anhydroglucitol attenuates cytokine release and protects mice with type 2 diabetes from inflammatory reactions.

1,5-anhydroglucitol (1,5-AG) decreases in diabetic patients and is used as a marker of glycemic control. Type 2 diabetic patients are susceptibile to lipopolysaccharides (LPS), which stimulate macrophages to release large quantities of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. This study examines the effects of 1,5-AG on lung inflammation induced by LPS and consequent systemic inflammation to determine whether the decrease of 1,5-AG concentration induces susceptibility to LPS. Before the challenge with LPS (1 mg/kg in vivo and 500 ng/ml in vitro), we pretreated db/db mice and RAW264.7 cells with 1,5-AG at 38.5 mg/kg and 500 microg/ml, respectively. The levels of IL-6, TNF-alpha, macrophage chemoattractant protein (MCP)-1 and IL-1beta in the serum and in the cell supernatants were measured. We also measured macrophage recruitment and the expression of inducible nitric oxide synthase (iNOS) in pulmonary tissues. We found that 1,5-AG attenuated serum cytokine release and protected db/db mice from LPS-induced pulmonary inflammation. In addition, 1,5-AG suppressed cytokine release and iNOS expression by suppressing Akt/NF-kB activity in RAW264.7 cells. These results suggest that 1,5-AG may be a mediator in, as well as marker for diabetes, and 1,5-AG intake may confer tolerance to LPS in patients with type 2 diabetes.

Galbeta1-6Gal, antigenic epitope which accounts for serological cross-reaction in diagnosis of Echinococcus multilocularis infection.

Parasitic helminths express various antigenic carbohydrates, which often account for serological cross-reactions. In serodiagnosis, it is essential to inspect cross-reactivity between the target parasite and other parasites in order to assess diagnostic performance. Our previous study showed that the Galbeta1-6Gal sequence was a common epitope between Echinococcus multilocularis (Em) and E. granulosus (Eg). Furthermore, compounds with this sequence from Fasciola hepatica (Fh) reportedly were recognized by sera with Eg infection. Our aim is to investigate whether this sequence is one of the widely common epitopes in many kinds of parasites. For various parasites, sera with Fh infection cross-reacted at the highest frequency (71.4%) against Em antigen. In patients with other parasitic infections, sera showed cross-reactions against Fh antigen bound to Em antigen with a high frequency (23.7%). Binding inhibition tests with commercial Galbeta1-6Gal disaccharide showed that Galbeta1-6Gal was the common epitope between not only Em, Eg and Fh, but also between various other parasites. Furthermore, the presence of the Galbeta1-6Gal epitope in Em antigen was confirmed by immunoblot testing with the specific antibody for this sequence. This study showed that the Galbeta1-6Gal sequence is one of the antigenic epitopes that accounts for serological cross-reactivity between Em and various other parasites.

First findings on the seroepidemiology of human paragonimosis at the anti-tuberculosis centre of Divo, Repubuc of Ivory Coast (West Africa).

An epidemiological study was carried out in 2004-2005 at the anti-tuberculosis centre of Divo (Ivory Coast) to collect sera from patients who consulted for tuberculosis suspicion and to estimate the seroprevalence of human paragonimosis in the context of a systematic screening. No Paragonimus egg was found in the stools and/or sputa of the 167 persons investigated. In contrast, 41 sera were ascertained with antibodies against Paragonimus africanus using ELISA testing. As the optical density (OD) values related to seropositive findings were found under 0.6 (the minimal OD to detect an active paragonimosis), the above antibody titres might originate from patients in chronic or in convalescent stages, or might result of cross reactions with trematodes. Concomitantly, dissection of local crabs (Callinectes marginatus) demonstrated the presence of Paragonimus metacercariae in six out of 34 examined. The parasite burdens in crabs ranged from two to 35 cysts with a mean diameter of 302 microm. In Ivory Coast, the locality of Divo must be considered an at-risk zone in reason of the presence of anti-Paragonimus antibodies in several human sera and the presence of infected crabs at the local market.

Suppressor of cytokine signalling 1 in lymphocytes regulates the development of intestinal inflammation in mice.

BACKGROUND AND AIMS: Imbalance between pro- and anti-inflammatory cytokines produced by intestinal T cells induces inflammatory bowel diseases (IBD). However, the importance of regulation of cytokine signalling in IBD has not been fully clarified. We have demonstrated that suppressor of cytokine signalling 1 (SOCS1) is expressed in inflamed tissues in an experimental colitis model. In the present study, we investigated the role of SOCS1 in colitis models to clarify the mechanism of IBD development. METHODS: Intestinal T cells in transgenic mice expressing high levels of SOCS1 in lymphocytes (SOCS1Tg mice) were characterised by flow cytometric analysis and cytokine production from intestinal T cells was determined by ELISA. 2,4,6-Trinitrobenzene sulphonic acid (TNBS) induced colitis was induced in SOCS1Tg mice and severity was compared with control littermates by measurement of survival rates. Intracellular signalling was assessed by western blotting analysis. RESULTS: SOCS1Tg mice developed colitis spontaneously with age. Young SOCS1Tg mice less than 15 weeks of age, before the onset of colitis, were susceptible to TNBS induced colitis. Intestinal T cells of SOCS1Tg mice showed increased interferon gamma and tumour necrosis factor alpha production and decreased transforming growth factor beta production. Expression of cytotoxic T lymphocyte associated antigen 4 (CTLA-4), a negative regulator of T cell activation, in SOCS1Tg mice was severely impaired at the protein level although mRNA levels of CTLA-4 in SOCS1Tg mice were comparable with those in control mice. CONCLUSIONS: Our data suggest that SOCS1 plays an important role in the regulation of colitis by controlling intestinal T cell activation mediated through CTLA-4 expression.

Bone marrow transplantation from unrelated donors for patients with adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATLL) is a highly aggressive haematological malignancy. More than 40 cases of ATLL treated by allogeneic bone marrow transplantation (BMT) from sibling donors have been reported, while there have been only a few cases of unrelated BMT for treatment of this disease. We began performing allogeneic BMT from unrelated donors in 1999 to improve the outcome of ATLL patients with no suitable sibling donors. Eight ATLL patients underwent unrelated BMT; five received the conventional conditioning regimen consisting of cyclophosphamide and total body irradiation, while three received a reduced-intensity preparative regimen. Two patients died due to encephalopathy of unknown aetiology on days 10 and 35, and one patient died due to progression of ATLL 25 months after BMT. Five patients are currently alive and disease-free at a median of 20 months after BMT. Proviral human T-lymphotropic virus type-I (HTLV-I) DNA load in peripheral blood mononuclear cells (PBMCs) was assessed in four cases before and after BMT. HTLV-I proviral DNA load was reduced significantly after transplantation. Unrelated BMT is feasible for treatment of ATLL. Further studies in a larger number of cases are required to determine the optimal conditioning regimen and stem cell source.

Evaluation of the antigenic similarities of adult-worm extracts from three Gnathostoma species, using sera from Mexican and Japanese patients with Gnathostoma infections.

The antigenic similarities of adult-worm extracts of Gnathostoma spinigerum, G. hispidum and G. doloresi, all of which are important food-borne parasites causing larva migrans in humans, were evaluated. The 40 sera used came from gnathostomiasis cases in Mexico, where G. binucleatum is endemic, or in Japan, where G. doloresi predominates. When used as the fixed antigens in microplate-ELISA, the adult-worm extracts from the three different species of Gnathostoma were found to have equal binding capacity to the Gnathostoma-specific IgG antibodies in the sera of the Mexican and Japanese patients. The correlation coefficients for the optical densities seen in the ELISA, between any two of the three Gnathostoma extracts, were all >0.900. The dose-response curves produced when four sera were tested, in the microplate-ELISA, against the three different Gnathostoma extracts were nearly identical, indicating that the antigens in each of the extracts had similar avidity. Furthermore, the results of competitive-inhibition ELISA indicated that the antigenic specificities of the three extracts were almost identical. An antigen of 40 kDa, which SDS-PAGE and immunoblot analysis revealed to be present in all three extracts, was recognized by the sera from the gnathostomiasis cases. When the sera were investigated by dot-blot ELISA, they also gave similar results whichever extract was used as the antigen source. It appears that, in the serodiagnosis of gnathostomiasis by microplate- or dot-ELISA, each of the three adult-worm extracts would be equally useful, regardless of the causative species of Gnathostoma.

The first human case in Mexico of conjunctivitis caused by the avian parasite, Philophthalmus lacrimosus.

Species of Philophthalmus parasitize primarily the eyes of wild and domestic birds. A variety of mammals, including humans, occasionally serve as the intermediate as well as the definitive hosts for this parasite, although human cases are extremely rare. Here, we report a case of human conjunctivitis caused by an infection with Philophthalmus sp. in Mexico. The patient was a 31-yr-old male who visited an ophthalmologist in Los Mochis, Sinaloa, Mexico, because of a foreign-body sensation in his left eye for 2 mo. A small live parasite was found in the connective tissue of the bulbar conjunctiva and was removed surgically under local anesthesia under ophthalmoscopic observation. The parasite was identified morphologically as Philophthalmus lacrimosus Braun, 1902. This is the first case of human philophthalmosis in Mexico and, to our knowledge, the first human case of P. lacrimosus infection in the world.