OBJECTIVES: To evaluate the protective effect of Schistosoma japonicum cystatin (rSj-Cystatin) in a mouse mode of "two-hit" sepsis. METHODS: Sixty male C57BL/6 mice randomized equally into sham-operated group, protein group, "two-hit" modeling group, and protein intervention group. In the former two groups, the mice received an intraperitoneal injection of 100 μL PBS followed by exposure of the cecum and then by intraperitoneal injection of 100 μL PBS or 25 μg rSj-Cystatin 30 min later; In the latter two groups, 100 μL PBS containing LPS (5 mg/kg) was injected intraperitoneally 24 h before cecal ligation and puncture (CLP), and 100 μL PBS or 25 μg rSj-Cystatin were injected 30 min after CLP. At 12 h after rSj-Cystatin treatment, 6 mice from each group were sacrificed for detection of TNF-α, IL-6, IL-10, TGF-β, iNOS and Arg-1 in the serum, spleen, liver, lung and kidney tissues using ELISA, for examinations of liver, lung and kidney pathologies with HE staining, and for analysis of CD3⁺CD4⁺CD25⁺Foxp3⁺ T cell percentage in the spleen using flow cytometry. The remaining mice were observed for general condition and 72-h survival. RESULTS: The 72-h survival rates in the 4 groups were 100%, 100%, 0% and 20%, respectively, showing significant differences between the latter two groups. The mouse models of "two-hit" sepsis exhibited obvious tissue pathologies and significant elevations of TNF-α and IL-6 in both the serum and tissue homogenate, which were significantly ameliorated by rSj-Cystatin treatment. Treatment with rSj-Cystatin also increased IL-10 and TGF-β levels and spleen CD3⁺CD4⁺CD25⁺Foxp3⁺ T cell percentage. The septic mouse models also showed increased iNOS levels in all the detected tissues and a decreased Arg-1 level in the kidney, and these changes were obviously improved by rSj-Cystatin treatment. CONCLUSIONS rSj-Cystatin has a protective effect against "two-hit" sepsis in mice by regulating the inflammatory microenvironment.
Animals ; Mice ; Sepsis/drug therapy* ; Male ; Schistosoma japonicum/chemistry* ; Mice, Inbred C57BL ; Cystatins/therapeutic use* ; Interleukin-10/metabolism* ; Interleukin-6/blood* ; Tumor Necrosis Factor-alpha/blood* ; Disease Models, Animal ; Transforming Growth Factor beta/metabolism*

OBJECTIVES: To explore the therapeutic mechanism of compound Centella asiatica formula (CCA) for alleviating Schistosoma japonicum (Sj)-induced liver fibrosis in mice. METHODS: The active components and targets of CCA were identified using the TCMSP database with cross-analysis of Sj-related liver fibrosis targets. A "drug-component-target-pathway-disease" network was constructed using Cytoscape 3.9.1. Functional enrichment analysis (GO/KEGG) was performed using DAVID. Molecular docking study was carried out to validate interactions between the core targets and the key compounds. For experimental validation of the results, 36 mice were divided into control group, Sj-infected model group, and CCA-treated groups. In the latter two groups, liver fibrosis was induced via abdominal infection with Sj cercariae for 8 weeks, followed by 8 weeks of daily treatment with CCA decoction or saline. Hepatic pathology of the mice was assessedwith HE and Masson staining, and hepatic expressions of collagen-I and collagen-III were detected using immunohistochemistry; serum IL-6 and TNF-α levels were determined with ELISA. Hepatic expressions of TLR4 and MyD88 proteins were analyzed with Western blotting. RESULTS: We identified a total of 107 bioactive CCA components and 791 targets, including 37 intersection targets linked to Sj-induced fibrosis. The core targets included TNF, TP53, JUN, MMP9, and CXCL8, involving the IL-17 signaling, lipid metabolism, TLR4/MyD88 axis, and cancer pathways. Molecular docking study confirmed strong binding affinity between quercetin (a primary CCA component) and TNF/TP53/JUN/MMP9. In Sj-infected mouse models, CCA treatment significantly attenuated hepatic inflammatory cell infiltration, reduced collagen-I and collagen-III deposition, improved tissue architecture, reduced serum IL-6 and TNF-α levels, and downregulated TLR4 and MyD88 expressions in the liver. CONCLUSIONS CCA mitigates Sj-induced liver fibrosis by targeting TNF, TP53, JUN, and MMP9 to modulate the TLR4/MyD88 pathway, thereby suppressing pro-inflammatory cytokine release, inhibiting hepatic stellate cell activation, reducing collagen deposition, and preventing granuloma formation in the liver.
Animals ; Toll-Like Receptor 4/metabolism* ; Mice ; Myeloid Differentiation Factor 88/metabolism* ; Schistosoma japonicum ; Liver Cirrhosis/parasitology* ; Schistosomiasis japonica ; Signal Transduction ; Molecular Docking Simulation ; Inflammation ; Centella/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism*

A 38-year-old female was admitted due to recurrent right upper quadrant (RUQ) abdominal pain. Three years prior to admission, the patient complained of a sudden onset of RUQ pain radiating to the right flank area. A month after the onset of pain, she consulted a physician, and she was advised to undergo endoscopy, but she did not comply. Three months after the consultation, an abdominal ultrasound was done, revealing a simple hepatic cyst. She then underwent aspiration of the cyst. However there was no resolution of the pain. A contrast-enhanced abdominal computed tomography (CT) scan revealed a recurrence of the hepatic cyst. Subsequently, she underwent laparoscopic fenestration of the cyst in liver segments VI, VII, and VIII. The biopsy results confirmed that the hepatic cyst was a hydatid cyst caused by Echinococcus granulosus. A month after the procedure, the abdominal pain recurred, and a repeat CT scan revealed another recurrence of the cyst. The patient was advised to undergo open surgery, but she did not consent. The patient denied taking anthelmintic drugs in the past. She did not experience any other symptoms—such as jaundice, nausea, vomiting, anorexia, or weight loss—along with the RUQ pain. She previously worked in Lebanon as a domestic helper for seven years and then moved to Taiwan, where she was employed for three years for the same work. She denied any direct exposure to dogs or sheep. On physical examination, she had a non-tender, firm, palpable mass in the RUQ area of the abdomen measuring 4x4 cm. The rest of the physical examination findings were unremarkable. A contrast-enhanced CT scan of the whole abdomen done two months prior to her admission showed multiple, well-defined, hypodense lesions in the right hepatic lobe. These lesions exhibited mildly enhancing walls and internal septations with rosette or honeycomb appearance. The two largest lesions seen in hepatic segments V and VII measured 8.6 x 6.1 x 5.2 cm and 9.5 x 8.5 x 7.4 cm, respectively (Figure 1). Similar hypodense lesions were seen in the right retroperitoneal space. At least two lesions were visible in the right perirenal space, measuring 8.8 x 6.1 x 6.5 cm and 6.1 x 5.9 x 3.8 cm, and at least two other lesions were visible in the right anterior pararenal space, within the region of the distal ascending colon, measuring 6.0 x 5.5 x 5.4 cm and 7.9 x 6.4 x 5.1 cm (Figure 2). At this point, we diagnosed the patient as having a recurrent hydatid cyst in the right hepatic lobe, with intraperitoneal extension in the right perirenal space and right anterior pararenal space, based on the CT scan findings. After securing medical clearance and administering mebendazole prophylactically at a dosage of 40 mg/kg/day, taken 3 times a day for 7 days prior to surgery, we aimed to sterilize the hydatid cysts and prevent surgical contamination.1 We did an exploratory laparotomy through a reverse L (Makuuchi) skin incision on the right upper abdominal quadrant (Figure 3). We subsequently performed a layered dissection down to the peritoneum. Intraoperatively, we noted adhesions at the posterior segments of the liver, right diaphragm and mid transverse colon. We then meticulously performed adhesiolysis. With extreme caution, we ensured to prevent any spillage of the cystic contents into adjacent structures and the abdominal cavity. Then, we covered the abdomen with abdominal packs soaked in 95% ethanol. Intraoperative liver ultrasonography revealed only one cystic lesion involving hepatic segments V, VI, and VII. The cyst was adherent to the right subdiaphragmatic area. We also noted multiple intraperitoneal cysts: one adherent to the right perirenal area measuring 8 x 6 cm, another adherent to the distal ascending colon and the mid transverse colon measuring 7 x 6 cm, and a third one non-adherent and floating in the anterior pararenal area measuring 4 x 4 cm. Before excising the intrahepatic mother cyst, we aspirated approximately 30-40 ml of cyst fluid and infused the cyst with an equivalent amount of 95% ethanol. After 15 minutes, we reaspirated the infused ethanol. We first carefully excised the intraperitoneal extensions of the cyst, (Figure 4) starting with the cyst in the distal ascending colon, then the cyst in the anterior pararenal area, and finally the right perirenal cyst, taking extreme caution to avoid rupturing the cyst. We then proceeded with the dissection of the hepatoduodenal ligament, where the portal triad is located, to provide access for our Pringle Maneuver. This step was followed by adhesiolysis at the right subdiaphragmatic area. Because of dense adhesions, there was a diaphragmatic injury, for which we performed phrenicorrhaphy. We then mobilized the right hepatic lobe through careful dissection of the triangular and coronary ligaments. The margins were marked under ultrasound guidance and the Pringle Maneuver was applied intermittently. We then carried out a parenchymal-sparing resection of segments V, VI, and VII. After securing hemostasis, we placed a Jackson Pratt drain in the subhepatic area. At the end of the surgery, we successfully resected hepatic segments V, VI, and VII, which contained an intrahepatic mother cyst with multiple spherical daughter cysts. Additionally, we excised three secondary cysts from the intraperitoneal area. Grossly, the excised specimen showed the right posterior segment of the liver with a surgically resected yellowish multiseptated cyst attached to it. This cyst has a fibrous rim and it contains several variable-sized daughter cysts (Figure 5). The excised intraperitoneal cyst from the distal ascending colon to mid transverse colon area (Figure 6), has a thick wall with a laminated external layer (Figure 7). The histopathological examination of the hydatid cyst showed E. granulosus protoscolices containing four acetabula and an armed rostellum with 30 to 36 hooks (Figure 8) . Sections of the liver showed liver parenchyma that was infiltrated by mononuclear cells. Adjacent to the liver parenchyma, the hydatid cyst wall contains an outer acellular laminated membrane, a germinal membrane, and remnants of the E. granulosus protoscolices, which resembled grains of sand (Figure 9 and 10). Postoperatively, the patient complained of dyspnea and tachypnea. On chest physical examination, we noted decreased breath sounds on the right. A chest x-ray done revealed a right-sided pneumothorax. Pneumothorax can occur during hepatectomy due to the escape of intraperitoneal carbon dioxide gas into the pleural cavity.2 We performed a chest tube thoracostomy insertion, and removed the tube after 5 days, once we observed full expansion of the patient’s right lung. We discharged the patient on the 9th postoperative day and prescribed oral antibiotics and pain relievers as home medications. Cystic echinococcosis (CE) or hydatid disease is a zoonotic parasitic disease caused by infection with the larval stage of the tapeworm E. granulosus. Dogs and other carnivores are definitive hosts, while sheep, cattle, and goats are intermediate hosts. Humans are accidental, intermediate hosts and become secondarily infected by ingesting food or water contaminated with eggs or gravid proglottids that are excreted in the definitive host’s feces. CE is endemic in North and East Africa, South America, China and Central Asia, and the Mediterranean countries.3 4 5 6 This disease has a low prevalence in Southeast Asia, particularly the Philippines.7 In our case, the patient probably acquired the infection when she worked in Lebanon, where echinococcosis is endemic. The liver and the lungs are the most common sites of the disease in humans. The concomitant occurrence of the cyst in the retroperitoneum or other intra-abdominal spaces is extremely rare,8 9 and it is associated with a high mortality and disability rate.10 Abdominal CT scan is a good imaging technique for visualizing wall calcifications in CE,11 and in our patient, enhancing walls and internal septations, which represent the walls of daughter cysts, were seen. Peritoneal seeding or cyst dissemination can occur due to a previous hepatic hydatid cyst surgery or after a spontaneous or traumatic rupture.12 These secondary cysts are formed from the liberated protoscolices caused by rupture of the daughter cysts.13 Although rare, when a cyst ruptures into the peritoneal cavity, it can lead to reactions ranging from mild hypersensitivity reaction to potentially fatal anaphylactic shock.14 A preoperative course of oral albendazole or mebendazole, as in the case of our patient’s medication, sterilizes the cysts and reduces their tension, thereby facilitating the surgery.15 16 It also kills most of the protoscolices within the hepatic hydatid cyst.17 Postoperatively, these drugs significantly reduce the risk of cyst recurrence.16 The prognosis of CE is generally good, and a complete cure is possible with total surgical excision without spillage.18 For hepatic echinococcosis, it is crucial to prioritize radical surgical resection in the initial surgery and preoperative anthelmintic therapy to minimize morbidity and recurrence often linked with conservative surgery.16 19
Cysts ; Echinococcus granulosus

OBJECTIVE: To evaluate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute kidney injury induced by acute liver failure and unravel the underlying mechanism, so as to provide insights into the clinical therapy of acute kidney injury. METHODS: Twenty-four male C57BL/6J mice at ages of 6 to 8 weeks were randomly divided into the normal control group, rSj-Cys control group, lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) model group and LPS/D-GaIN + rSj-Cys treatment group, of 6 mice each group. Mice in the LPS/D-GaIN group and LPS/D-GaIN + rSj-Cys group were intraperitoneally injected with LPS (10 μg/kg) and D-GaIN (700 mg/kg), and mice in the LPS/D-GaIN + rSj-Cys group were additionally administered with rSj-Cys (1.25 mg/kg) by intraperitoneal injection 30 min post-modeling, while mice in the rSj-Cys group were intraperitoneally injected with rSj-Cys (1.25 mg/kg), and mice in the normal control group were injected with the normal volume of PBS. All mice were sacrificed 6 h post-modeling, and mouse serum and kidney samples were collected. Serum creatinine (Cr) and urea nitrogen (BUN) levels were measured, and the pathological changes of mouse kidney specimens were examined using hematoxylin-eosin (HE) staining. Serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were detected using enzyme-linked immunosorbent assay (ELISA), and the expression of inflammatory factors and pyroptosis-related proteins was quantified in mouse kidney specimens using immunohistochemistry. In addition, the expression of pyroptosis-related proteins and nuclear factor-kappa B (NF-κB) signaling pathway-associated proteins was determined in mouse kidney specimens using Western blotting assay. RESULTS: HE staining showed no remarkable abnormality in the mouse kidney structure in the normal control group and the rSj-Cys control group, and renal tubular injury was found in LPS/D-GaIN group, while the renal tubular injury was alleviated in LPS/D-GaIN+rSj-Cys treatment group. There were significant differences in serum levels of Cr (F = 46.33, P < 0.001), BUN (F = 128.60, P < 0.001), TNF-α (F = 102.00, P < 0.001) and IL-6 (F = 202.10, P < 0.001) among the four groups, and lower serum Cr [(85.35 ± 32.05) μmol/L], BUN [(11.90 ± 2.76) mmol/L], TNF-α [(158.27 ± 15.83) pg/mL] and IL-6 levels [(56.72 ± 4.37) pg/mL] were detected in the in LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (all P values < 0.01). Immunohistochemical staining detected significant differences in TNF-α (F = 24.16, P < 0.001) and IL-10 (F = 15.07, P < 0.01) expression among the four groups, and lower TNF-α [(106.50 ± 16.57)%] and higher IL-10 expression [(91.83 ± 5.23)%] was detected in the LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (both P values < 0.01). Western blotting and immunohistochemistry detected significant differences in the protein expression of pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (F = 24.57 and 30.72, both P values < 0.001), IL-1β (F = 19.24 and 22.59, both P values < 0.001) and IL-18 (F = 16.60 and 19.30, both P values < 0.001) in kidney samples among the four groups, and lower NLRP3, IL-1β and IL-18 expression was quantified in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (P values < 0.05). In addition, there were significant differences in the protein expression of NF-κB signaling pathway-associated proteins p-NF-κB p-P65/NF-κB p65 (F = 71.88, P < 0.001), Toll-like receptor (TLR)-4 (F = 45.49, P < 0.001) and p-IκB/IκB (F = 60.87, P < 0.001) in mouse kidney samples among the four groups, and lower expression of three NF-κB signaling pathway-associated proteins was determined in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (all P values < 0.01). CONCLUSIONS rSj-Cys may present a protective effect against acute kidney injury caused by acute liver failure through inhibiting inflammation and pyroptosis and downregulating the NF-κB signaling pathway.
Mice ; Male ; Animals ; Interleukin-10 ; Tumor Necrosis Factor-alpha/genetics* ; NF-kappa B/therapeutic use* ; Interleukin-18/therapeutic use* ; Schistosoma japonicum/metabolism* ; Interleukin-6/therapeutic use* ; Lipopolysaccharides/therapeutic use* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Mice, Inbred C57BL ; Acute Kidney Injury/drug therapy* ; Liver Failure, Acute ; Cystatins/therapeutic use*

OBJECTIVE: To evaluate the molluscicidal activity of surfactin against Oncomelania hupensis, so as to provide the experimental basis for use of Bacillus for killing O. hupensis. METHODS: O. hupensis snails were collected from schistosomiasisendemic foci of Wuhu City on September 2022, and Schistosoma japonicum-infected snails were removed. Then, 60 snails were immersed in surfactin at concentrations of 2, 1, 0.5, 0.25, 0.125 mg/mL and 0.062 5 mg/mL for 24, 48, 72 hours at 26 °C, while ultrapure water-treated snails served as controls. The median lethal concentration (LC₅₀) of surfactin against O. hupensis snails was estimated. O. hupensis snails were immersed in surfactin at a concentration of 24 h LC₅₀ and ultrapure water, and then stained with propidium iodide (PI). The PI uptake in haemocyte was observed in O. hupensis snails using fluorescence microscopy. RESULTS: The mortality of O. hupensis was 5.0% following immersion in surfactin at a concentration of 0.062 5 mg/mL for 24 h, and the mortality was 100.0% following immersion in surfactin at a concentration of 2 mg/mL for 72 h, while no snail mortality was observed in the control group. There were significant differences in the mortality of O. hupensis in each surfactin treatment groups at 24 (χ² = 180.150, P < 0.05), 48 h (χ² = 176.786, P < 0.05) and 72 h (χ² = 216.487, P < 0.05), respectively. The average mortality rates of O. hupensis were 38.9% (140/360), 62.2% (224/360) and 83.3% (300/360) 24, 48 h and 72 h post-immersion in surfactin, respectively (χ² = 150.264, P < 0.05), and the 24, 48 h and 72 h LC₅₀ values of surfactin were 0.591, 0.191 mg/mL and 0.054 mg/mL against O. hupensis snails. Fluorescence microscopy showed more numbers of haemocytes with PI uptake in 0.5 mg/mL surfactintreated O. hupensis snails than in ultrapure water-treated snails for 24 h, and there was a significant difference in the proportion of PI uptake in haemocytes between surfactin-and ultrapure water-treated snails (χ² = 6.690, P < 0.05). CONCLUSIONS Surfactin is active against O. hupensis snails, which may be associated with the alteration in the integrity of haemocyte membrane.
Animals ; Molluscacides/pharmacology* ; Snails ; Schistosoma japonicum ; Lethal Dose 50 ; Water

Echinococcosis is a zoonotic parasitic disease caused by infection with Echinococcus species. As the drug of first choice for treatment of echinococcosis, albendazole suffers from problems of large doses and remarkable adverse reactions in clinical therapy. Development of novel drugs against echinococcosis is of urgent need. Recently, great advances have been achieved in the research on traditional Chinese medicine for treatment of echinococcosis. This review summarizes the progress of researches on traditional Chinese medicine for treatment of echinococcosis, aiming to provide insights into development of anti-echinococcosis drugs.
Animals ; Humans ; Medicine, Chinese Traditional ; Echinococcosis/parasitology* ; Albendazole/therapeutic use* ; Zoonoses ; Echinococcus multilocularis

OBJECTIVE: To investigate the spatial distribution characteristics of the prevalence of advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody, and to examine the correlation between the prevalence of advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody in Hunan Province in 2020, so as to provide insights into advanced schistosomiais control in the province. METHODS: The epidemiological data of schistosomiasis in Hunan Province in 2020 were collected, including number of permanent residents in survey villages, number of advanced schistosomiasis patients, number of residents receiving serological tests and number of residents seropositive for anti-Schistosoma antibody, and the prevalence advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody were descriptively analyzed. Village-based spatial distribution characteristics of prevalence advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody were identified in Hunan Province in 2020, and the correlation between the revalence advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody was examined using Spearman correlation analysis. RESULTS: The prevalence of advanced schistosomiasis was 0 to 2.72% and the seroprevalence of anti-Schistosoma antibody was 0 to 20.25% in 1 153 schistosomiasis-endemic villages in Hunan Province in 2020. Spatial clusters were identified in both the prevalence of advanced schistosomiasis (global Moran's I = 0.416, P < 0.01) and the seroprevalence of anti-Schistosoma antibody (global Moran's I = 0.711, P < 0.01) in Hunan Province. Local spatial autocorrelation analysis identified 98 schistosomiasis-endemic villages with high-high clusters of the prevalence of advanced schistosomiasis, 134 endemic villages with high-high clusters of the seroprevalence of anti-Schistosoma antibody and 36 endemic villages with high-high clusters of both the prevalence of advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody in Hunan Province. In addition, spearman correlation analysis showed a positive correlation between the prevalence of advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody (r_s = 0.235, P < 0.05). CONCLUSIONS There were spatial clusters of the prevalence of advanced schistosomiasis and seroprevalence of anti-Schistosoma antibody in Hunan Province in 2020, which were predominantly located in areas neighboring the Dongting Lake. These clusters should be given a high priority in the schistosomiasis control programs.
Animals ; Humans ; Prevalence ; Seroepidemiologic Studies ; Schistosomiasis/epidemiology* ; Schistosoma ; Spatial Analysis ; Antibodies, Helminth ; China/epidemiology*

OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ² = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ² = 11.20, P < 0.05) and fluorescent RAA assay (χ² = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.
Animals ; Clonorchis sinensis/genetics* ; Metacercariae/genetics* ; Recombinases ; Fresh Water ; Fishes ; DNA ; Fish Diseases/diagnosis*

OBJECTIVE: To establish a surveillance and early warning index system for schistosomiasis transmission risk along the middle and lower reaches of the Yangtze River basin, so as to provide insights into creation of a sensitive and highly efficient surveillance and early warning system for schistosomiasis. METHODS: National and international publications, documents, laws and regulations pertaining to schistosomiasis control were retrieved with keywords including schistosomiasis, surveillance, early warning and control interventions from 2008 to 2022, and a thematic panel discussion was held to preliminarily construct surveillance and early warning index system for schistosomiasis transmission risk along the middle and lower reaches of the Yangtze River basin. The index system was then comprehensively scored and screened using the Delphi method, and the weight of each index was determined using analytic hierarchy process and the modified proportional allocation method. In addition, the credibility of the Delphi method was evaluated using positive coefficient, authority coefficient, degree of concentration and degree of coordination of experts. RESULTS: Following two rounds of expert consultation, a surveillance and early warning index system for schistosomiasis transmission risk in endemic areas along the middle and lower reaches of the Yangtze River basin was preliminarily constructed, including 3 primary indicators, 9 secondary indicators and 41 tertiary indicators. The normalized weights of primary indicators epidemics, natural and social factors and comprehensive control were 0.639 8, 0.145 6 and 0.214 6, respectively, and among all secondary indicators, snail status (0.321 3) and schistosomiasis prevalence (0.318 5) had the highest combined weights, while social factors had the lowest combined weight (0.030 4). Of all tertiary indicators, human egg-positive rate (0.041 9), number of acute schistosomiasis cases (0.041 5), number of stool-positive bovine and sheep (0.041 1), and prevalence of Schistosoma japonicum in free-ranging livestock (0.041 1) had the highest combined weights. During two rounds of consultation, the positive coefficient of experts was both 100%, and the authority coefficient was both 0.9 and greater, while the coordination coefficients were 0.338 to 0.441 and 0.426 to 0.565 (χ² = 22.875 to 216.524, both P values < 0.05). CONCLUSIONS The established surveillance and early warning index system for schistosomiasis transmission risk along the middle and lower reaches of the Yangtze River basin is of great scientific values and authority, which may provide insights into construction of the sensitive and highly efficient surveillance and early warning system for schistosomiasis in the context of low prevalence and low intensity of infection in China.
Animals ; Cattle ; Humans ; Sheep ; Rivers ; Schistosomiasis/prevention & control* ; Schistosoma japonicum ; China/epidemiology* ; Snails

OBJECTIVE: To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China. METHODS: Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup. RESULTS: The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1). CONCLUSIONS Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.
Animals ; Paragonimus/genetics* ; Brachyura/genetics* ; Cyclooxygenase 1/genetics* ; Phylogeny ; China/epidemiology* ; Sequence Analysis, DNA ; Paragonimiasis