Development and evaluation of an immunochromatography-based point-of-care test kit for a rapid diagnosis of human cysticercosis.

Human cysticercosis is a life-threatening zoonotic disease caused by infection with larvae (cysticerci) of the pork tapeworm, Taenia solium. This can affect the nervous system causing chronic headache and intracranial hypertension, potentially leading to epileptic seizures and paralysis. The disease is found in developing countries, especially in Southeast and South Asia, Sub-Saharan Africa, and Central and South America where porcine cysticercosis is endemic and people have a habit of eating undercooked pork. An immunochromatography-based test (ICT) kit, using T. solium cyst fluid as antigen, was manufactured to detect anti-T. solium IgG antibodies in human serum. To evaluate the kit, we used 187 serum samples including 24 from proven/confirmed cysticercosis cases, 133 from cases with other parasitosis and 30 healthy controls. Diagnostic efficiencies were calculated. The sensitivity, specificity, and accuracy were 83.3%, 92.0%, and 90.9%, respectively. Moreover, the ICT was positive before treatment but became negative after treatment, implying that this kit is also useful for follow-up monitoring post-treatment. In conclusion, we have successfully developed and present preliminary evaluation of an easy-to-handle rapid diagnostic tool for human cysticercosis in the form of an ICT platform using as antigen fluid from T. solium cysticerci.

Intron Regions as Genetic Markers for Population Genetic Investigations of Opisthorchis viverrini sensu lato and Clonorchis sinensis.

Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called "O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts.

Evaluation of total immunoglobulin G and subclass antibodies in an enzyme-linked immunosorbent assay for serodiagnosis of human amebic liver abscess.

Background: Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA. Methods: A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients (n = 14), healthy individuals (n = 40), and patients with other diseases (n = 276). Results: ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8-100.0] and 97.8% specificity 95% CI [95.5-99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8-100.0]) and a slightly higher specificity (99.1% 95% CI [97.3-99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar's test; p > 0.05), with a concordance of 98.2%, represented by a Cohen's kappa of 0.83 (p < 0.001), indicating almost perfect agreement. Conclusion: ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis.

Rapid label-free detection of cholangiocarcinoma from human serum using Raman spectroscopy.

Cholangiocarcinoma (CCA) is highly prevalent in the northeastern region of Thailand. Current diagnostic methods for CCA are often expensive, time-consuming, and require medical professionals. Thus, there is a need for a simple and low-cost CCA screening method. This work developed a rapid label-free technique by Raman spectroscopy combined with the multivariate statistical methods of principal component analysis and linear discriminant analysis (PCA-LDA), aiming to analyze and classify between CCA (n = 30) and healthy (n = 30) serum specimens. The model's classification performance was validated using k-fold cross validation (k = 5). Serum levels of cholesterol (548, 700 cm-1), tryptophan (878 cm-1), and amide III (1248,1265 cm-1) were found to be statistically significantly higher in the CCA patients, whereas serum beta-carotene (1158, 1524 cm-1) levels were significantly lower. The peak heights of these identified Raman marker bands were input into an LDA model, achieving a cross-validated diagnostic sensitivity and specificity of 71.33% and 90.00% in distinguishing the CCA from healthy specimens. The PCA-LDA technique provided a higher cross-validated sensitivity and specificity of 86.67% and 96.67%. To conclude, this work demonstrated the feasibility of using Raman spectroscopy combined with PCA-LDA as a helpful tool for cholangiocarcinoma serum-based screening.

An Innovative Test for the Rapid Detection of Specific IgG Antibodies in Human Whole-Blood for the Diagnosis of Opisthorchis viverrini Infection.

Chronic human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis can last for decades and cause liver and biliary diseases, including life-threatening pathology prior to cholangiocarcinoma (CCA). CCA generally has a poor prognosis. Serological diagnosis can support parasitological examination in diagnosing disease and screening for the risk of CCA. Here, we present an improved and innovative lateral flow immunochromatographic test (ICT) kit that uses whole-blood samples (WBS) rather than serum to diagnose human opisthorchiasis, which also successfully diagnosed human clonorchiasis. This ICT includes a soluble worm extract of O. viverrini adults and colloidal-gold-labeled conjugates of the IgG antibody to evaluate the diagnostic values with simulated WBS (n = 347). Simulated WBS were obtained by the spiking infection sera with red blood cells. The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy for detecting opisthorchiasis were 95.5%, 87.0%, 80.5%, 97.2%, and 90.1%, respectively. For clonorchiasis, these findings were 85.7%, 87.0%, 53.6%, 97.2%, and 86.8%, respectively. Combined for both diseases, they were 93.2%, 87.0%, 84.0%, 94.6%, and 89.6%, respectively. The ICT kit can possibly replace the ICT platforms for antibody detection in serum samples in field surveys in remote areas where sophisticated equipment is not available.

TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection.

Angiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

Rapid assessment of Opisthorchis viverrini IgG antibody in serum: A potential diagnostic biomarker to predict risk of cholangiocarcinoma in regions endemic for opisthorchiasis.

BACKGROUND: Opisthorchiasis is caused by an infection with fish-borne liver flukes of the genus Opisthorchis. Opisthorchiasis frequently leads to chronic inflammation in the biliary tract and is classified as a group 1 biological carcinogen by the International Agency for Research on Cancer: a definitive risk for cholangiocarcinoma (CCA). METHODS: We used the rapid immunochromatographic test (ICT) to detect anti-Opisthorchis viverrini IgG and IgG4 subclass antibodies in sera of patients with CCA. The ICT kits were developed based on soluble antigens excreted and secreted by O. viverrini adult worms. RESULTS: ICT indicated sera was positive for IgG and IgG4 antibodies, respectively, in 22 (61.1%) and 15 (41.6%) participants of the 36 study participants diagnosed with CCA (P > 0.05). Our study also included groups with other cancers and with liver cirrhosis, where the IgG ICT and IgG4 ICT kits were 27.7% (13/47) and 25.5% (12/47) positive, respectively (P > 0.05). Neither total the IgG ICT nor the IgG4 ICT yielded positive results in a control group of 20 healthy participants. Moreover, the percentage positivity rate using the ICT for total IgG between the CCA group and the other cancers and liver cirrhosis group was significantly different (P < 0.05). By contrast, no significant difference between these groups was apparent in the ICT for IgG4 antibody. The CCA group was 6.53 times more likely to have positive anti-O. viverrini IgG antibody (odds ratio 6.53, P < 0.001) and 3.27 times more likely to have positive anti-O. viverrini IgG4 antibody (odds ratio 3.27, P = 0.010) than the non-CCA group. CONCLUSION: This information is of potential value for the development of a diagnostic biomarker to predict risk for O. viverrini infection-associated CCA.

Comparative assessment of immunochromatographic test kits using somatic antigens from adult Opisthorchis viverrini and IgG and IgG4 conjugates for serodiagnosis of human opisthorchiasis.

Chronic infections of humans with Opisthorchis viverrini and Clonorchis sinensis spanning decades may lead to life-threatening pathology prior to cholangiocarcinoma (CCA), which usually has a poor prognosis. Serological tools can support the parasitological examination in clinical diagnosis and support screening for risk of CCA. We developed novel immunochromatographic test kits using a soluble, somatic tissue extract of adult O. viverrini worms as an antigen and colloidal gold-labeled conjugates of IgG and IgG4 antibodies, and evaluated the diagnostic values of both the OvSO-IgG and OvSO-IgG4 kits. For diagnosis of human opisthorchiasis individually, the diagnostic sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals in the OvSO-IgG kit were 86.6% (78.9-92.3), 89.5% (84.2-93.5), 82.9% (74.8-89.2), and 91.9% (87.0-95.4), respectively, while the 75% (65.9-82.7), 98.4% (95.5-99.7), 96.6% (90.3-99.3), and 87% (81.7-91.2), respectively, for the OvSO-IgG4 kit at the prevalence of infection of 37.1%. Twenty-three (76.7%) and 14 (46.7%) of 30 clonorchiasis sera showed positive reactivity with the OvSO-IgG and OvSO-IgG4 kits, respectively. There was 84.1% (κ-value = 0.649) concordance between the two kits, which was statistically significant (p < 0.001). Both ICT kits can be employed as quick and easy point-of-care diagnostic tools, and hence, the OvSO-IgG and OvSO-IgG4 kits can support expanded capacity for clinical diagnosis of human opisthorchiasis and clonorchiasis. These kits may find utility in large-scale surveys in endemic areas where there are limited sophisticated medical facilities or capacity.

An Unusual Case of Gastric Gnathostomiasis Caused by Gnathostoma spinigerum Confirmed by Video Gastroscopy and Morphological and Molecular Identification.

Human gnathostomiasis is a harmful foodborne parasitic infection caused by nematodes of the genus Gnathostoma. Here, we report an unusual case of gastric gnathostomiasis seen in a hospital in Thailand along with the clinical characteristics, treatment, and outcome. A 39-year-old man presented with complaints of epigastric pain, dizziness, and history of passing dark, tarry stools for 2 days. The patient had a history of consuming raw freshwater fish. Supplementary differential diagnosis was performed via rapid serological testing, and presence of the causative agent was confirmed based on video gastroscopy, morphology of the removed parasite, and molecular identification. After its surgical removal from the stomach, the parasite was morphologically identified as Gnathostoma species. Molecular identification was performed via DNA extraction from the recovered worm, and amplification and sequencing of the second internal transcribed spacer (ITS2) region and partial cytochrome c oxidase subunit I (cox1) gene. The ITS2 and cox1 sequences were consistent with those of Gnathostoma spinigerum. Clinicians in endemic areas should therefore be aware of the rare clinical manifestations and use of supplementary serological tests to facilitate early diagnosis and treatment of gastric gnathostomiasis.

High prevalence of opisthorchiasis in rural populations from Khammouane Province, central Lao PDR: serological screening using total IgG- and IgG4-based ELISA.

BACKGROUND: Human opisthorchiasis, caused by Opisthorchis viverrini, is a public health problem in Southeast Asia and a major risk factor for cholangiocarcinoma. In Lao PDR, seroprevalence and the relationship between the number of O. viverrini eggs in infected people and specific antibody responses are still unknown. We evaluated and compared parasitological and serological screening methods in the community in an endemic area of opisthorchiasis in Lao PDR. METHODS: Seroprevalence of O. viverrini-specific total IgG and IgG4 antibodies and their relationships with O. viverrini egg intensities were evaluated in Khammouane Province, central Lao PDR, using ELISA and a modified formalin ethyl-acetate concentration technique (FECT). RESULTS: FECT stool examination revealed O. viverrini eggs in 70.3% (90/128) of individuals (95% CI 61.6 to 78.1%) while ELISA (based on total IgG and on IgG4 antibodies to O. viverrini) found 98.4% (95% CI 94.5 to 99.8%) and 89.8% (95% CI 83.3 to 94.5%) of sera, respectively. There was a positive and significant correlation between numbers of O. viverrini eggs per gram and levels of both IgG (R2=0.168, p<0.001) and IgG4 (R2=0.219, p<0.001) antibodies. CONCLUSIONS: A high prevalence of human opisthorchiasis in Lao PDR was found using a new platform, serological screening in the community. This points to a need for sustainable control of this liver fluke infection.

Monosexual Cercariae of Schistosoma japonicum Infection Protects Against DSS-Induced Colitis by Shifting the Th1/Th2 Balance and Modulating the Gut Microbiota.

Inflammatory bowel disease (IBD)-related inflammation is closely associated with the initiation and progression of colorectal cancer. IBD is generally treated with 5-aminosalicylic acid and immune-modulating medication, but side effects and limitations of these therapies are emerging. Thus, the development of novel preventative or therapeutic approaches is imperative. Here, we constructed a dextran sodium sulphate (DSS)-induced IBD mouse model that was infected with monosexual Schistosoma japonicum cercariae (mSjci) at day 1 or administered dexamethasone (DXM) from days 3 to 5 as a positive control. The protective effect of mSjci on IBD mice was evaluated through their assessments of their clinical signs, histopathological lesions and intestinal permeability. To uncover the underlying mechanism, the Th1/Th2 balance and Treg cell population were also examined. Additionally, the alterations in the gut microbiota were assessed to investigate the interaction between the mSjci-modulated immune response and pathogenic microbiome. Mice treated with DSS and mSjci showed fewer IBD clinical signs and less impaired intestinal permeability than DSS-treated mice. Mechanistically, mSjci modulated the Th1/Th2 balance by repressing IFN-γ production, promoting IL-10 expression and enhancing the Treg subset population. Moreover, mSjci notably reshaped the structure, diversity and richness of the gut microbiota community and subsequently exerted immune-modulating effects. Our findings provide evidence showing that mSjci might serve as a novel and effective protective strategy and that the gut microbiota might be a new therapeutic target in IBD.

Development of immunochromatographic device as a point-of-care tool for serodiagnosis of human strongyloidiasis cases.

Human strongyloidiasis is an important gastrointestinal disease with an estimated 30 to 100 million people infected. Prevalence is generally underestimated since many infections are asymptomatic, and traditional diagnostic tests based on parasitological examination of stool samples are not adequately sensitive. Serological tests are useful and supportive but are still only available in a reference research setting. We made an immunochromatographic test (ICT) kit for rapid serodiagnosis of human strongyloidiasis. The antigen used in the ICT kit was extracted from larvae of Strongyloides stercoralis. Diagnostic efficacy of the kit was evaluated using human serum samples from strongyloidiasis patients, healthy persons, and those with other parasitoses. When using a cutoff level of 0.5 or above, the diagnostic sensitivity, specificity, and positive and negative predictive values at the prevalence of infection of 34.4%, were 93.3%, 83.7%, 76.7%, and 95.6%, respectively. This ICT kit is easy to use at the point-of-care and a result can be obtained in 15 min. Sophisticated instruments and highly trained staff are not required. It can be used in several diagnostic and public-health settings, e.g., prevalence surveys in endemic areas, confirmation and monitoring of cure post-treatment, diagnosis and screening of infected but asymptomatic individuals, and populations "at risk" for hyperinfection syndrome or disseminated strongyloidiasis if they are given immunosuppressive treatment for other conditions.

Abdominal angiostrongyliasis can be diagnosed with a immunochromatographic rapid test with recombinant galactin from Angiostrongylus cantonensis

Angiostrongylus costaricensis is the causative agent of abdominal angiostrongyliasis, a zoonotic infection that may produce severe eosinophilic enterocolitis or hepatitis in humans. Parasites are usually not released in stools and serology has an important role in diagnosis. Since cross-reactivity is demonstrated between A. costaricensis and another metastrongylid worm, A. cantonensis, we tested heterologous recombinant galectin as a probe in an immunochromatographic rapid diagnostic test (ICT-RDT) for detection of anti-A. costaricensis antibodies. Almost all (11/12) positive control sera from A. costaricensis infected patients were positive at ICT RDT. These are preliminary indications that r-galectin ICT-RDT is useful for diagnosing A. costaricensis infection.

Development of an Immunochromatographic Point-of-Care Test for Serodiagnosis of Opisthorchiasis and Clonorchiasis.

Chronic infections with the food-borne liver flukes, Opisthorchis viverrini or Clonorchis sinensis, associate with cholangiocarcinoma, bile duct cancer, which generally has a poor prognosis. We have produced a rapid and simple immunochromatographic test (ICT) kit for the diagnosis of opisthorchiasis and clonorchiasis by the detection of IgG antibodies in human infection sera. Sera from volunteers with proven opisthorchiasis and several other parasitic diseases and from healthy controls were evaluated for the presence of liver fluke infection-specific antibodies using a preparation of excretory-secretory antigen from adult stage O. viverrini absorbed onto ICT strips. Diagnostic values were compared with an ELISA. The diagnostic sensitivity, specificity, and positive and negative predictive values of the ELISA were 100%, 98.3%, 97.9%, and 100%, whereas those for the ICT were 94.6%, 91.2%, 89.7%, and 95.4%, respectively. There was 91.7% concordance between the ICT with ELISA, and differences in performance between the tests were not statistically significant (P > 0.05). Twenty-seven of 30 (90%) of the clonorchiasis sera also were positive by ICT. This new ICT provides a facile, rapid test for point-of-care testing tool, which can be used at the bedside without the need for sophisticated equipment. Moreover, the ICT can be anticipated to supplement stool examination as a screening tool in the clinic for the diagnosis of opisthorchiasis and clonorchiasis, and in addition, it may be useful in screens of populations at risk of liver fluke infection-associated cholangiocarcinoma.

Application of Recombinant Angiostrongylus cantonensis Galectin-2 Protein for Serodiagnosis of Human Angiostrongyliasis by Immunoblotting.

Angiostrongyliasis is a foodborne disease caused by a zoonotic nematode, Angiostrongylus cantonensis, which produces eosinophilic meningitis or meningoencephalitis (EOM) in humans. Definitive diagnosis is rarely possible because worms are almost never recovered from patients. Human disease can be diagnosed by clinical symptoms and serological tests. Presently, diagnosis is performed by serological detection of antibodies against specific somatic antigens (molecular mass 29-31 kDa) extracted from female worms. The life cycle of A. cantonensis must be maintained in the laboratory to provide a source of this diagnostic antigen. Here, we cloned and expressed recombinant A. cantonensis galectin-2 (rAcGal2) corresponding to a 31-kDa antigenic peptide. Recombinant protein was purified and used in immunoblot tests, which showed reactions with human serum panels consisting of six confirmed angiostrongyliasis and 24 clinically diagnosed cases of EOM-associated with angiostrongyliasis, 160 samples from patients with other parasitic infections, and 30 samples from normal healthy subjects. Accuracy, sensitivity, specificity, and positive and negative predictive values were 95.0%, 93.3%, 95.3%, 75.7%, and 98.9%, respectively. The test was nonreactive with sera of human gnathostomiasis and cysticercosis, two diseases that could present similar neurological symptoms. Recombinant AcGal2 has potential as a diagnostic antigen and could replace native parasite antigens in further development of an angiostrongyliasis serodiagnostic test kit.

Development and evaluation of a rapid diagnostic immunochromatographic device to detect antibodies in sera from intestinal capillariasis cases.

Intestinal capillariasis, a fish-borne nematodiasis, is an important emerging zoonotic disease. Patients present clinical symptoms of borborygmus chronic diarrhea, intermittent abdominal pain, weight loss, and several degrees of painless lower-leg edema. Death may occur in cases of misdiagnosis and improper treatment. Diagnosis is difficult because of the atypical clinical symptoms and diagnostic confusion with diarrhea caused by gastrointestinal cancer, opportunistic infections in human immunodeficiency virus patients, and hyperthyroidism. In addition, parasite eggs are not always found in stool examination. Serology can provide a supportive diagnostic tool. We have produced a rapid and simple immunochromatographic test (ICT) kit for diagnosis of intestinal capillariasis by detection of diagnostic antibodies in human sera. Serum samples from healthy volunteers and patients with proven intestinal capillariasis and other parasitic diseases were evaluated with the Trichinella spiralis larval extract antigen absorbed ICT strips. The diagnostic sensitivity, specificity, and positive and negative predictive values were 100, 96.6, 90.2, and 100%, respectively. The ICT kit is simple and rapid to use and can supplement stool examination in clinical diagnosis of intestinal capillariasis. The test can be completed in 15 min without a need for any sophisticated instruments or reagents.

Rapid identification of nine species of diphyllobothriidean tapeworms by pyrosequencing.

The identification of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea) that infect humans and intermediate/paratenic hosts is extremely difficult due to their morphological similarities, particularly in the case of Diphyllobothrium and Spirometra species. A pyrosequencing method for the molecular identification of pathogenic agents has recently been developed, but as of yet there have been no reports of pyrosequencing approaches that are able to discriminate among diphyllobothriidean species. This study, therefore, set out to establish a pyrosequencing method for differentiating among nine diphyllobothriidean species, Diphyllobothrium dendriticum, Diphyllobothrium ditremum, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium stemmacephalum, Diplogonoporus balaenopterae, Adenocephalus pacificus, Spirometra decipiens and Sparganum proliferum, based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as a molecular marker. A region of 41 nucleotides in the cox1 gene served as a target, and variations in this region were used for identification using PCR plus pyrosequencing. This region contains nucleotide variations at 12 positions, which is enough for the identification of the selected nine species of diphyllobothriidean tapeworms. This method was found to be a reliable tool not only for species identification of diphyllobothriids, but also for epidemiological studies of cestodiasis caused by diphyllobothriidean tapeworms at public health units in endemic areas.

Strongyloides stercoralis diagnostic polypeptides for human strongyloidiasis and their proteomic analysis.

Human strongyloidiasis is a deleterious gastrointestinal disease mainly caused by Strongyloides stercoralis infection. Strongyloides stercoralis is a soil-transmitted helminthiasis that is distributed around the globe. Although definitive diagnosis is carried out through the detection of parasite objects in human stool samples, the development of reliable immunological assays is an important alternative approach for supportive diagnosis. We characterized the two sensitive and specific bands of S. stercoralis filariform larvae that reacted with human strongyloidiasis sera based on immunoblot analysis. Serum samples obtained from strongyloidiasis patients showed a sensitivity of 90 and 80 % at the approximate molecular mass of 26 and 29-kDa polypeptide bands, respectively. The reactive specificity of the 26-kDa band was 76.5 % while for the 29-kDa band was 92.2 %. Proteomic analysis identified the 26-kDa band protein was 14-3-3 protein zeta, while the 29-kDa band protein was ADP/ATP translocase 4. The results provided a basic framework for further studies regarding the potential of the S. stercoralis recombinant antigen to become a leading to diagnostic tool.

Identification in immuno-electron microscopy of solitary multi-polar peripheral neurons of adult Opisthorchis viverrini by antibody against rat diacylglycerol kinase / Identificación por microscopía imuno-electrónica de las neuronas periféricas multipolares solitarias del Opisthorchis viverrini adulto mediante anticuerpo contra la diacilglicerol kinasa de ratas

By utilizing the antibody for rat DGKz a substantial number of immunopositive cells were found in the OV (Opisthorchis viverrini). The immunopositive cells appeared solitarily and they were distributed rather symmetrically to the longitudinal axis of the OV. Some of them were located in close proximity to internal organs such as uterus, ovary, testes, vitelline glands and guts. The immunostained cells extended tapering processes horizontally or obliquely to the OV longitudinal axis. In immuno-electron microscopy, the immunopositive cells were characterized by intensely immunostained mitochondria and weakly immunostained cytoplasm and immunonegative chromatin-poor nucleus. Vacuoles of various sizes without the immunoreactivity were also contained in the cells. Thin cellular processes without the immunoreactivity were found to enclose thinly the entire surfaces of the immunostained cells and processes, and they were in continuity with the interstitial partition-like processes which contained nuclei and aggregation of microfibrils at some distance from the cytoplasmic envelopes. The present finding suggests the possibility that the immunostained cells were peripheral neurons enveloped by peripheral glia and that the glia are of mesenchymal origin because of their cytoplasmic continuity to the interstitial partition-like processes. The motor or sensory nature of the neurons remains to be elucidated.

Mediante el uso del anticuerpos DGK para rata se determinó un número considerable de células inmunopositivas en el Opisthorchis viverrini (OV). Las células inmunopositivas aparecían solitarias y se distribuían simétricamente al eje longitudinal de la OV. Algunas estaban ubicadas en las proximidades de los órganos internos como el útero, ovarios, testículos, glándulas vitelinas e intestino. Las células inmunoteñidas extendían sus procesos horizontalmente u oblicuamente al eje longitudinal de la OV. Por microscopía inmunoelectrónica, las células inmunopositivas se caracterizaron por presentar mitocondrias intensamente teñidas, citoplasma con tinción débil e inmunonegatividad en núcleos pobres en cromatina. También se observó en las células, vacuolas de diversos tamaños sin inmunorreactividad. Se encontraron procesos celulares sin inmunorreactividad para cerrar finamente todas las superficies de las células y procesos, y se continuaron con los procesos de partición intersticiales que contenían núcleos y agregación de microfibrillas a cierta distancia de las envolturas citoplásmicas. El presente hallazgo sugiere la posibilidad de que las células inmunoteñidas son neuronas periféricas envueltas por glia periférica y que la glía presenta origen mesenquimal debido a su continuidad citoplasmática con los procesos de partición intersticiales. La naturaleza motora o sensorial de las neuronas aún no se ha dilucidado.

INTRAOSSEOUS PROLIFERATIVE SPARGANOSIS PRESENTING AS A PATHOLOGICAL FRACTURE: A CASE REPORT AND REVIEW OF THE LITERATURE.

We report a case of 63-year-old male, who presented with pathological fracture of left distal humerus 3 weeks previously. The radiographic findings showed an ill-defined permeative osteolytic lesion of the left distal humerus. Incisional biopsy and debridement was done; pathological examination revealed a folded cestode larva with calcareous corpuscles in the bone and soft tissue, and increased eosinophils. IgG antibody tests for sparganosis were positive. The patient refused to have surgery for internal fixation and placement of an endoprosthesis.