Detection of Gnathostoma spinigerum larva in the brain with complete follow-up after surgical treatment of human neurognathostomiasis.

Human gnathostomiasis is a food-borne zoonotic helminthic infection widely reported in Latin America, Asia, and Southeast Asia. Consuming raw, or under-cooked fresh-water fish is the leading cause of this helminthic infection, which is clinically characterized by signs of inflammation, itching sensation, or irritation with migratory swelling. Neurological symptoms resulting from neurognathostomiasis vary, and there is scant information due to the rareness of patient brain samples. This study aimed to demonstrate the first evidence of human neurognathostomiasis by the detection of Gnathostoma spinigerum larva in patient's brain during craniotomy, supported by histopathological, immunological and proteomic evidence. Clinical symptoms were obtained from medical history and physical examination with laboratory investigations, including magnetic resonance imaging (MRI), left temporal craniotomy, histopathology of brain tissue, and Western blot analysis, were performed to elucidate the causative pathogens for diagnosis. In addition, the host-parasite interaction of the parasite invading the patient's brain was characterized through proteomics. Histopathology revealed worms with the characteristic cuticular spines of G. spinigerum which were detected and identified. These histopathological findings were consistent with a positive Western blot showing a 24-kDa reactive-band for gnathostomiasis. Proteomic analysis revealed the presence of G. spinigerum serpin and serine protease in the patient's serum. Moreover, the leucine-rich alpha-2-glycoprotein was indicated as a systemic biomarker of early brain injury related to invasion by G. spinigerum. Therefore, our study provides the initial evidence of human neurognathostomiasis due to G. spinigerum larval invasion along with successful craniotomy and proven larval detection including complete follow-up, and the disease prognosis after surgical treatment.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

BACKGROUND: Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces. METHODS: To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. RESULTS: The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10-1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. CONCLUSIONS: The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Prevalence of Strongyloides in Southeast Asia: a systematic review and meta-analysis with implications for public health and sustainable control strategies.

BACKGROUND: Strongyloidiasis, caused by the nematodes Strongyloides stercoralis and Strongyloides fuelleborni, is estimated to affect over 600 million individuals worldwide. The disease is endemic in Southeast Asia, where a warm-humid climate and socio-economic conditions maintain the parasite's life cycle and transmission. However, the current diagnostic methods may not be sufficiently sensitive, suggesting that the true prevalence of strongyloidiasis could be seriously underestimated in this. This study aims to determine the prevalence of strongyloidiasis in Southeast Asia through a systematic review and meta-analysis and to discuss the implications of the estimated prevalence on diagnostic approaches and control strategies. METHODS: Following PRISMA guidelines, we conducted a systematic literature search in PubMed and Google Scholar databases to identify studies reporting Strongyloides prevalence data in the 11 Southeast Asian countries up to December 2022. A random effects model was employed to estimate the pooled prevalence of S. stercoralis at both regional and country levels. RESULTS: Out of 3722 articles identified, 224 met our inclusion criteria. For S. stercoralis specifically, we found 187 articles, of which 52.4% were from Thailand. All Southeast Asian countries, except Brunei, had at least one study on Strongyloides prevalence. The estimated pooled prevalence of S. stercoralis regionally was 12.7% (95% CI 10.70-14.80%), ranging from 0.4 to 24.9% at the country level. Cambodia had the highest pooled prevalence (24.9%, 95% CI 15.65-35.38%), followed by Lao PDR (16.5%, 95% CI 9.50-24.95%). Moreover, we obtained a pooled prevalence of 10% (95% CI 7.06-13.52%) in a group comprising immigrants, workers, and veterans from Southeast Asian countries. S. stercoralis infects various host types, including nonhuman primates, domestic dogs and cats, rodents, and transport carriers such as cockroaches and vegetables. CONCLUSIONS: A high prevalence of strongyloidiasis in Southeast Asia was revealed, highlighting the importance of the region's ongoing research, surveillance, and control efforts. Factors contributing to the strongyloidiasis transmission include the role of animal hosts, the impact of global connectivity, and the significance of the co-endemicity of other Strongyloides species. Based on these findings, a multi-pronged One-Health approach is essential for sustainable intervention and control.

Assessment of an Immuno-Diagnostic Method for Hookworm-Related Cutaneous Larva Migrans Using Crude Extracts of Ancylostoma caninum.

People can become infected with cutaneous larva migrans (CLM) through skin penetration by the infective zoonotic larvae of hookworms. Few studies have investigated CLM's immunodiagnosis, and the existing studies were limited to crude somatic or excretory/secretory antigens (Ags) from adult worms. Here, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate and diagnose hwCLM by detecting immunoglobulin (Ig)E, IgG, and IgG subclasses 1-4 (IgG1-4) against the somatic Ag of adult Ancylostoma caninum checkerboard titrations of adult A. caninum worm extract. Pooled serum controls were immunocharacterized using an indirect ELISA. The IgG1-4 and IgE results were unsatisfactory; however, the use of total IgG achieved results comparable to those of immunoblotting. Thus, we continued to analyze the IgG-ELISA using serum samples from patients with hwCLM and heterologous infections as well as from healthy controls. The sensitivity and excellent specificity of the total IgG-ELISA were 93.75% and 98.37%, respectively, and its positive and negative predictive values were 75% and 99.67%, respectively. Antibodies from five cases of angiostrongyliasis, gnathostomiasis, and dirofilariasis cross-reacted with the somatic Ag of adult A. caninum. This new assay can adequately serodiagnose hwCLM when combined with clinical features and/or histological examination.

Transcriptome profiling of male and female Ascaris lumbricoides reproductive tissues.

BACKGROUND: Ascaris lumbricoides causes human ascariasis, the most prevalent helminth disease, infecting approximately 1 billion individuals globally. In 2019 the global disease burden was estimated to be 754,000 DALYs and resulted in 2090 deaths. In the absence of a vaccination strategy, treatment of ascariasis has relied on anthelminthic chemotherapy, but drug resistance is a concern. The propensity for reinfection is also a major challenge to disease control; female worms lay up to 200,000 eggs daily, which contaminate surrounding environments and remain viable for years, resulting in high transmission rates. Understanding the molecular mechanisms of reproductive processes, including control of egg production, spermatogenesis, oogenesis and embryogenesis, will drive the development of new drugs and/or vaccine targets for future ascariasis control. METHODS: Transcriptome profiles of discrete reproductive and somatic tissue samples were generated from adult male and female worms using Illumina HiSeq with 2 × 150 bp paired-end sequencing. Male tissues included: testis germinal zone, testis part of vas deferens, seminal vesicle and somatic tissue. Female tissues included: ovary germinal zone, ovary part of the oviduct, uterus and somatic tissue. Differentially expressed genes (DEGs) were identified from the fragments per kilobases per million reads (FPKM) profiles. Hierarchical analysis was performed to identify tissue-specific genes. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to identify significant terms and pathways for the DEGs. RESULTS: DEGs involved in protein phosphorylation and adhesion molecules were indicated to play a crucial role in spermatogenesis and fertilization, respectively. Those genes associated with the G-protein-coupled receptor (GPCR) signaling pathway and small GTPase-mediated signal transduction pathway play an essential role in cytoskeleton organization during oogenesis. Additionally, DEGs associated with the SMA genes and TGF-ß signaling pathway are crucial in adult female embryogenesis. Some genes associated with particular biological processes and pathways that were identified in this study have been linked to defects in germline development, embryogenesis and reproductive behavior. In the enriched KEGG pathway analysis, Hippo signaling, oxytocin signaling and tight junction pathways were identified to play a role in Ascaris male and female reproductive systems. CONCLUSIONS: This study has provided comprehensive transcriptome profiles of discrete A. lumbricoides reproductive tissue samples, revealing the molecular basis of these functionally important tissues. The data generated from this study will provide fundamental knowledge on the reproductive biology of Ascaris and will inform future target identification for anti-ascariasis drugs and/or vaccines.

Strongyloides stercoralis: A Neglected but Fatal Parasite.

Strongyloidiasis is a disease caused by Strongyloides stercoralis and remains a neglected tropical infection despite significant public health concerns. Challenges in the management of strongyloidiasis arise from wide ranging clinical presentations, lack of practical high sensitivity diagnostic tests, and a fatal outcome in immunocompromised hosts. Migration, globalization, and increased administration of immunomodulators, particularly during the COVID-19 era, have amplified the global impact of strongyloidiasis. Here, we comprehensively review the diagnostic tests, clinical manifestations, and treatment of strongyloidiasis. The review additionally focuses on complicated strongyloidiasis in immunocompromised patients and critical screening strategies. Diagnosis of strongyloidiasis is challenging because of non-specific presentations and low parasite load. In contrast, treatment is simple: administration of single dosage ivermectin or moxidectin, a recent anthelmintic drug. Undiagnosed infections result in hyperinfection syndrome and disseminated disease when patients become immunocompromised. Thus, disease manifestation awareness among clinicians is crucial. Furthermore, active surveillance and advanced diagnostic tests are essential for fundamental management.

Automatic recognition of parasitic products in stool examination using object detection approach.

Background: Object detection is a new artificial intelligence approach to morphological recognition and labeling parasitic pathogens. Due to the lack of equipment and trained personnel, artificial intelligence innovation for searching various parasitic products in stool examination will enable patients in remote areas of undeveloped countries to access diagnostic services. Because object detection is a developing approach that has been tested for its effectiveness in detecting intestinal parasitic objects such as protozoan cysts and helminthic eggs, it is suitable for use in rural areas where many factors supporting laboratory testing are still lacking. Based on the literatures, the YOLOv4-Tiny produces faster results and uses less memory with the support of low-end GPU devices. In comparison to the YOLOv3 and YOLOv3-Tiny models, this study aimed to propose an automated object detection approach, specifically the YOLOv4-Tiny model, for automatic recognition of intestinal parasitic products in stools. Methods: To identify protozoan cysts and helminthic eggs in human feces, the three YOLO approaches; YOLOv4-Tiny, YOLOv3, and YOLOv3-Tiny, were trained to recognize 34 intestinal parasitic classes using training of image dataset. Feces were processed using a modified direct smear method adapted from the simple direct smear and the modified Kato-Katz methods. The image dataset was collected from intestinal parasitic objects discovered during stool examination and the three YOLO models were trained to recognize the image datasets. Results: The non-maximum suppression technique and the threshold level were used to analyze the test dataset, yielding results of 96.25% precision and 95.08% sensitivity for YOLOv4-Tiny. Additionally, the YOLOv4-Tiny model had the best AUPRC performance of the three YOLO models, with a score of 0.963. Conclusion: This study, to our knowledge, was the first to detect protozoan cysts and helminthic eggs in the 34 classes of intestinal parasitic objects in human stools.

Evaluation of indirect-ELISA using eluted antigens from Trichinella spiralis muscle larvae for diagnosis of swine trichinellosis.

Trichinellosis is caused by Trichinella spiralis muscle larvae (TsML), which is transmitted to human when they eat infected raw or undercooked meat. T. spiralis infection is detected by an enzyme-linked immunosorbent assay (ELISA) using excretory-secretory antigens (ESAg); however, the preparation of ESAg is challenging, and yields are low, which hampers screening efforts. In this study, crude somatic antigens (CSAg) of TsML with molecular weights (MWs) of 43, 79 and 101 kDa have been identified in swine trichinellosis sera with less cross-reaction with uninfected sera and other parasitic infected sera. After that, the CSAg at MWs of 43, 79 and 101 kDa (TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively) were isolated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted antigens were analyzed by IgG-ELISA for sensitivity and specificity, and specific antigens from the three regions were identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The sensitivity of IgG-ELISA using the three eluted antigens was 100% with specificities of 97.77%, 95.54%, 90.63% and for TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively. The LC-MS-MS results of immunomics showed that 18/20 spots of the antigens with MWs of 43, 79, and 101 kDa represent 11 different proteins identified. TsCSAg-43 showed the highest specificity, indicating that the specific proteins identified, including 45 kDa antigen-trichina [fragment], DNA topoisomerase 2-alpha antigen targeted by protective antibodies, and a conserved hypothetical protein (gi339234223), should be developed and produced in large volumes for further immunodiagnostic studies.

Survival of immature pre-adult Gnathostoma spinigerum in humans after treatment with albendazole.

Human gnathostomiasis is a food-borne zoonotic helminthic infection widely reported in Latin America, Asia and Southeast Asia, particularly in Thailand. There are increasing reports of the parasite in countries where it is not endemic. A study of the survival drug-treated immature stage (STIM) of Gnathostoma spinigerum recovered from infected patients focused on their integument surface using scanning electron microscopy (SEM). STIM displayed a specific, characteristic head bulb, with a pair of large thick equal-sized trilobulated lips in the centre. Cephalic spines had eight transverse rows on the head bulb with single-ended tips curved posteriorly. Body cuticular spines on the anterior half of the STIM were not sharp-pointed but distributed more densely, with multi-dentated-cuticular spines, irregularly arranged in a lining pattern of velvety cuticular folds. The length of cuticular spines increased caudally. The size of spines became gradually smaller, and numbers decreased towards the posterior end. Spines were still widely dispersed posteriorly as their density dropped. The morphology of STIM of G. spinigerum are described in detail for the first time. These specimens showed structural adaptation based on changes on integument surfaces, probably to protect against damage induced by the toxic effects of albendazole.

Evaluation of immunodiagnostic tests for human gnathostomiasis using different antigen preparations of Gnathostoma spinigerum larvae against IgE, IgM, IgG, IgG1-4 and IgG1 patterns of post-treated patients.

OBJECTIVES: The aims of the study were two-fold: (1) antigen (Ag) preparation and evaluation of three antigens of Gnathostoma spinigerum infective larvae (GsL3), crude somatic antigen (CSAg), excretory-secretory antigen (ESAg) and partially purified antigens (namely P1Ag, P2Ag and P3Ag) to differentiate IgE, IgG, IgG1-4 and IgM for human gnathostomiasis diagnosis; and (2) application of the selected ELISA for following up stored sera of patients treated with ivermectin (IVM) and albendazole (ABZ). METHODS: Different antigens were analysed by antibodies of gnathostomiasis cases, other parasite infections and healthy controls using indirect ELISA to differentiate IgE, IgG, IgG1-4 and IgM. Then, prominent antigen and immunoglobulin were used in antibody predictions of gnathostomiasis cases treated with albendazole or ivermectin. RESULTS: Sensitivity of all evaluated ELISAs: IgM-, IgG-, IgG1- and IgG4-ELISA, was 100%. IgM-ELISA with CSAg and P3Ag exhibited the highest specificity of 99%. IgG-ELISA with P2Ag resulted in the highest specificity of 92.3%. IgG1-ELISA with P2Ag and P3Ag showed excellent results with 100% specificity. Finally, P2Ag evaluated IgG1 of the followed-up cases with ABZ and IVM. Decreasing antibody IgG1 levels were mostly found in both treatments at Month 9 and long follow-up was over 12 months. A Gnathostoma worm was extracted from each two treated patients. CONCLUSIONS: Using IgG1-ELISA against P2Ag and P3Ag gave excellent results with 100% sensitivity and specificity. These tests can be an alternative to immunoblotting for gnathostomiasis. IgG1 decreased at least 9 months in most cases, so long-term treatment should be performed over 1 year.

Co-occurrence of Angiostrongylus malaysiensis and Angiostrongylus cantonensis DNA in cerebrospinal fluid: Evidence from human eosinophilic meningitis after ingestion of raw snail dish in Thailand.

Angiostrongylus cantonensis, the main causative agent of human neuroangiostrongyliasis, is a food-borne parasitic zoonosis, particularly in Southeast Asia and Mainland China. Angiostrongylus malaysiensis, a cryptic species, has not been unequivocally identified as a causative agent for human angiostrongyliasis. Here, we investigated a local incidence of human angiostrongyliasis in Kalasin Province, northeastern part of Thailand. Field and laboratory investigations, clinical symptoms, and treatment of the disease are also discussed. Five sera and three cerebrospinal fluid samples were taken from each patient who displayed clinical symptoms of mild or severe headache without neck stiffness after ingesting a local dish containing Pila virescens. With molecular evidence using PCR and DNA sequencing approaches, we confirmed the presence of A. malaysiensis and A. cantonensis DNA in the patient samples. In addition, P. virescens and Pomacea canaliculata collected in the vicinity were also examined for the existence of angistrongylid larvae. The rate of infection in the snail population was 33.3% (18 infection out of 54 examined), with A. cantonensis as the predominant species. Notably, two snails were found to be co-infected with both A. malaysiensis and A. cantonensis. This discovery comes after several years of suspicion that it could be a zoonotic pathogen. Therefore, our findings are important for public health and clinical diagnosis since clinicians are not aware of the zoonotic potential of A. malaysiensis in humans.

Risk factors and prevalence of taeniasis among the Karen people of Tha Song Yang District, Tak Province, Thailand.

Taeniasis remains a prevalent public health problem in Thailand. National helminthiasis surveys report only the incidence of Taenia spp. eggs. The ability to differentiate Taenia species using morphological and molecular techniques is vital for epidemiological surveys. This study detected taeniasis carriers and other helminthic infections by Kato's thick smear technique and identified the Taenia species by multiplex PCR. The study subjects were the ethnic Karen people in Tha Song Yang District, Tak Province, Thailand, bordering Myanmar. In total, 983 faecal samples from villagers were examined for helminthiases. Interview-based questionnaires were used to gather information on possible risk factors for infection. The prevalence of helminth infections was 42.7% (420/983), including single (37.3%, 367/983) and mixed infections (5.4%, 53/983). The most common infection (19.23%, 189/983) was Ascaris lumbricoides, whereas taeniasis carriers comprised 2.8% (28/983). Multiplex PCR of Cox1 was used for species identification of Taenia tapeworms, eggs, or both in 22 taeniasis carriers. Most of the parasites (20 cases) were Taenia solium, with two cases of Taenia saginata. Taenia saginata asiatica was not found in the villagers examined. The analysis of 314 completed questionnaires showed that a statistically significant (p < 0.05) risk of taeniasis was correlated with being male, a history of being allowed to forage during childhood, a history of seeing tapeworm proglottids, and a history of raw or undercooked pork consumption. Health education programmes must seek to reduce and prevent reinfection in these communities.

TITLE: Facteurs de risque et prévalence de la téniase chez les Karens du district de Tha Song Yang, province de Tak, Thaïlande. ABSTRACT: La téniase reste un problème de santé publique répandu en Thaïlande. Les enquêtes nationales sur les helminthiases ne rapportent que l'incidence des œufs de Taenia spp. La capacité de différencier les espèces de Taenia à l'aide de techniques morphologiques et moléculaires est vitale pour les enquêtes épidémiologiques. Cette étude a détecté des porteurs de téniase et d'autres infections helminthiques par la technique de frottis épais de Kato et a identifié les espèces de Taenia par PCR multiplex. Les sujets de l'étude étaient les Karens du district de Tha Song Yang, province de Tak, Thaïlande, à la frontière du Myanmar. Au total, 983 échantillons de matières fécales provenant de villageois ont été examinés pour les helminthiases. Des questionnaires basés sur des entretiens ont été utilisés pour recueillir des informations sur les facteurs de risque possibles d'infection. La prévalence des helminthes était de 42,7 % (420/983), dont des infections uniques (37,3 %, 367/983) et mixtes (5,4 %, 53/983). L'infection la plus courante (19,23 %, 189/983) était Ascaris lumbricoides, tandis que les porteurs de téniase représentaient 2,8 % (28/983). La PCR multiplexe de Cox1 a été utilisée pour l'identification des adultes ou des oeufs de Taenia, ou des deux, chez 22 porteurs de téniase. La plupart des parasites (20 cas) étaient Taenia solium, avec deux cas de Taenia saginata. Taenia saginata asiatica n'a pas été trouvé chez les villageois examinés. L'analyse de 314 questionnaires a montré qu'un risque statistiquement significatif (p < 0,05) de téniase était en corrélation avec le fait d'être un homme, et des antécédents d'avoir été autorisé à ramasser sa nourriture pendant l'enfance, d'avoir vu des proglottis de ténia et de consommation de porc cru ou insuffisamment cuit. Les programmes d'éducation sanitaire doivent chercher à réduire et à prévenir la réinfection dans ces communautés.

Improved Detection of Intestinal Helminth Infections with a Formalin Ethyl-Acetate-Based Concentration Technique Compared to a Crude Formalin Concentration Technique.

Intestinal helminth infections are the most prevalent neglected tropical diseases, predominantly affecting rural and marginalised populations. The mainstay of diagnosis is the microscopic examination of faecal samples to detect parasites in the form of eggs, larvae and cysts. In an effort to improve the standard of care, the comparative accuracy in detecting helminth infections of the hitherto used formalin-based concentration method (FC) was compared to a previously developed formalin ethyl-acetate-based concentration technique (FECT), prior to the systematic deployment of the latter at a research and humanitarian unit operating on the Thailand-Myanmar border. A total of 693 faecal samples were available for the comparison of the two diagnostic methods. The FECT was superior in detecting hookworm, Trichuris trichiura and small liver flukes. Interestingly, there was no significant difference for Ascaris lumbricoides, possibly due to the high observed egg density. Despite the minor increase in material cost and the fact that the FECT is somewhat more time consuming, this method was implemented as the new routine technique.

Case Report: The First Direct Evidence of Gnathostoma spinigerum Migration through Human Lung.

Gnathostomiasis is a helminthic infection caused by the third-stage larvae of nematodes of the genus Gnathostoma. The life cycle in humans starts with an enteric phase, with the worm perforating the gastric or intestinal mucosa to reach the peritoneal cavity and migrating through the human body. Subsequent penetration through the diaphragm may produce pleuropulmonary symptoms. We herein present a previously healthy 56-year-old Thai man from Southern Thailand who was an ex-smoker presented with chronic dry cough progressing to hemoptysis after consuming grilled swamp eels and freshwater fish. Chest computed tomography showed consolidation at the lingular segment, and the differential diagnosis was primary lung cancer and pulmonary tuberculosis. The lung tissue biopsied during bronchoscopy displayed segments of organisms with the phenotypic characteristics of Gnathostoma spp., and abundant eosinophils were seen in the alveolar tissue. Gnathostoma spinigerum infection was confirmed by a Western blot assay for G. spinigerum-specific 24-kDa reactive band. The patient received albendazole, and a follow-up chest radiograph revealed improvement in the consolidation in the lung and reduction in hemoptysis. We report the first direct evidence including pathology and immunohistochemistry of Gnathostoma invasion via the human lung, with clinical and radiographic presentations mimicking either malignancy or chronic infection.

Intestinal parasites in rural communities in Nan Province, Thailand: changes in bacterial gut microbiota associated with minute intestinal fluke infection.

Gastrointestinal helminth infection likely affects the gut microbiome, in turn affecting host health. To investigate the effect of intestinal parasite status on the gut microbiome, parasitic infection surveys were conducted in communities in Nan Province, Thailand. In total, 1047 participants submitted stool samples for intestinal parasite examination, and 391 parasite-positive cases were identified, equating to an infection prevalence of 37.3%. Intestinal protozoan species were less prevalent (4.6%) than helminth species. The most prevalent parasite was the minute intestinal fluke Haplorchis taichui (35.9%). Amplicon sequencing of 16S rRNA was conducted to investigate the gut microbiome profiles of H. taichui-infected participants compared with those of parasite-free participants. Prevotella copri was the dominant bacterial operational taxonomic unit (OTU) in the study population. The relative abundance of three bacterial taxa, Ruminococcus, Roseburia faecis and Veillonella parvula, was significantly increased in the H. taichui-infected group. Parasite-negative group had higher bacterial diversity (α diversity) than the H. taichui-positive group. In addition, a significant difference in bacterial community composition (ß diversity) was found between the two groups. The results suggest that H. taichui infection impacts the gut microbiome profile by reducing bacterial diversity and altering bacterial community structure in the gastrointestinal tract.

Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain).

BACKGROUND: Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1). RESULTS: Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion. CONCLUSIONS: SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Transcriptomic analysis of male and female Schistosoma mekongi adult worms.

BACKGROUND: Schistosoma mekongi is one of five major causative agents of human schistosomiasis and is endemic to communities along the Mekong River in southern Lao People's Democratic Republic (Laos) and northern Cambodia. Sporadic cases of schistosomiasis have been reported in travelers and immigrants who have visited endemic areas. Schistosoma mekongi biology and molecular biology is poorly understood, and few S. mekongi gene and transcript sequences are available in public databases. RESULTS: Transcriptome sequencing (RNA-Seq) of male and female S. mekongi adult worms (a total of three biological replicates for each sex) were analyzed and the results demonstrated that approximately 304.9 and 363.3 million high-quality clean reads with quality Q30 (> 90%) were obtained from male and female adult worms, respectively. A total of 119,604 contigs were assembled with an average length of 1273 nt and an N50 of 2017 nt. From the contigs, 20,798 annotated protein sequences and 48,256 annotated transcript sequences were obtained using BLASTP and BLASTX searches against the UniProt Trematoda database. A total of 4658 and 3509 transcripts were predominantly expressed in male and female worms, respectively. Male-biased transcripts were mostly involved in structural organization while female-biased transcripts were typically involved in cell differentiation and egg production. Interestingly, pathway enrichment analysis suggested that genes involved in the phosphatidylinositol signaling pathway may play important roles in the cellular processes and reproductive systems of S. mekongi worms. CONCLUSIONS: We present comparative transcriptomic analyses of male and female S. mekongi adult worms, which provide a global view of the S. mekongi transcriptome as well as insights into differentially-expressed genes associated with each sex. This work provides valuable information and sequence resources for future studies of gene function and for ongoing whole genome sequencing efforts in S. mekongi.

ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand.

BACKGROUND: Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. METHODS: To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 µg/ml, and anti-human IgG diluted at 1:4000. RESULTS: The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). CONCLUSIONS: Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis.

Apoptosis and necroptosis of mouse hippocampal and parenchymal astrocytes, microglia and neurons caused by Angiostrongylus cantonensis infection.

BACKGROUND: Angiostrongylus cantonensis has been the only parasite among Angiostrongylidae to cause human central nervous system infection characterized by eosinophilic meningitis or meningoencephalitis. The mechanism of the extensive neurological impairments of hosts caused by A. cantonensis larvae remains unclear. The aim of the present study was to investigate apoptosis, necroptosis and autophagy in the brains of mice infected with A. cantonensis, which will be valuable for better understanding the pathogenesis of angiostrongyliasis cantonensis. METHODS: Functional and histological neurological impairments of brain tissues from mice infected with A. cantonensis were measured by the Morris water maze test and haematoxylin and eosin (H&E) staining, respectively. The transcriptional and translational levels of apoptosis-, necroptosis- and autophagy-related genes were quantified by quantitative real-time polymerase chain reaction (RT-PCR), and assessed by western blot and immunohistochemistry (IHC) analysis. Apoptotic and necroptotic cells and their distributions in infected brain tissues were analysed by flow cytometry and transmission electron microscopy (TEM). RESULTS: Inflammatory response in the central nervous system deteriorated as A. cantonensis infection evolved, as characterized by abundant inflammatory cell infiltration underneath the meninges, which peaked at 21 days post-infection (dpi). The learning and memory capacities of the mice were significantly decreased at 14 dpi, indicating prominent impairment of their cognitive functions. Compared with those of the control group, the mRNA levels of caspase-3, -4, -6, and RIP3 and the protein levels of caspase-4, cleaved caspase-3, cleaved caspase-6, RIP3, and pRIP3 were obviously elevated. However, no changes in the mRNA or protein levels of FADD, Beclin-1 or LC3B were evident, indicating that apoptosis and necroptosis, but not autophagy, occurred in the brain tissues of mice infected with A. cantonensis. The quantitative RT-PCR, western blot, IHC, flow cytometry and TEM results further revealed the apoptotic and necroptotic microglia, astrocytes and neurons in the parenchymal and hippocampal regions of infected mice. CONCLUSIONS: To our knowledge, we showed for the first time that A. cantonensis infection causes the apoptosis and necroptosis of microglia and astrocytes in the parenchymal and hippocampal regions of host brain tissues, further demonstrating the pathogenesis of A. cantonensis infection and providing potential therapeutic targets for the management of angiostrongyliasis.