Objective To investigate the mechanism of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) in regulation of immune responses against Cryptosporidium parvum infections in intestinal epithelial cells. Methods Human colorectal cell line HCT-8 was infected with C. parvum oocysts (oocysts to cell ratio of 2:1) for 0, 4, 8, 12, 24, 36 and 48 h. Then, total protein was extracted from cells, and the relative STING protein expression was determined using Western blotting. Cell models of cGAS and STING knockdown were generated using small interfering RNA (siRNA). The relative expression of cGAS, STING, TANK-binding kinase 1 (TBK-1), p-TBK-1, interferon regulatory factor (IRF3), p-IRF3, nuclear factor-κB (NF-κB), p-NF-κB, interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) was determined using Wester blotting in HCT-8 cells 24 h post-infection, and the apoptosis of HCT-8 cells was detected using flow cytometry before and after STING knockdown. In addition, the relative expression level of C. parvum 18S mRNA was quantified using quantitative fluorescent PCR (qPCR) before and after STING knockdown. Statistical analysis of the data was conducted using the independent samples t-test. Results Western blotting assay determined that the relative STING protein expression was 3.000 ± 0.743, 2.756 ± 0.847, 2.397 ± 0.701, 3.645 ± 0.306, 3.773 ± 0.471, 3.982 ± 0.468 in HCT-8 cells 4, 8, 12, 24, 36 and 48 h post-infection with C. parvum, which was all higher than in uninfected cells (0 h) (1.000 ± 0.039) (t = 4.655, 3.587, 3.448, 14.870, 10.160, 11.000; all P < 0.05). The relative expression of cGAS, STING, p-NF-κB/NF-κB and TNF-α, p-TBK-1/TBK-1, p-IRF3/IRF3, IFN-β proteins was 1.024 ± 0.093, 1.042 ± 0.160, 1.060 ± 0.108, 0.665 ± 0.297, 0.929 ± 0.207, 0.740 ± 0.104 in C. parvum infected HCT-8 cells with STING knockdown, which was all lower than in infected cells (1.757 ± 0.332, 2.329 ± 0.336, 1.522 ± 0.230, 1.339 ± 0.088, 1.332 ± 0.036) (t = 10.25, 3.360, 6.231, 3.949, 3.159, 9.362; all P < 0.05), and the relative expression of cGAS and STING proteins was 0.771 ± 0.038 and 0.696 ± 0.094 in C. parvum infected HCT-8 cells with cGAS knockdown, which was all lower than in infected cells (1.231 ± 0.074, 1.238 ± 0.023) (t = 9.608, 9.674; both P < 0.05). Flow cytometry detected a lower apoptotic rate in C. parvum infected HCT-8 cells with STING knockdown [(18.90 ± 0.75)%] than in infected cells [(23.72 ± 2.55)%] (t = 3.141, P < 0.05), and qPCR assay quantified higher relative expression of C. parvum 18S mRNA in C. parvum infected HCT-8 cells with STING knockdown (1.335 ± 0.037) than in infected cells (1.002 ± 0.071) (t = 7.195, P < 0.05). Conclusion cGAS-STING contributes to host immune responses against C. parvum infections via activation of TBK1 and NF-κB signaling pathways.

Objective To investigate the changes in the gene expression profile of human monocyte THP-1 cells overexpressing Toxoplasma gondii rhoptry protein 16 (ROP16) using transcriptome sequencing, and to screen immune response-related driver genes. Methods Human monocytic THP-1 cells were seeded onto 96-well plates. Cells in the overexpression group was transfected with ROP16 overexpression lentivirus for 72 h, and cells in the control group were treated with an equal volume of culture medium. The efficiency of cell transfection was checked under a fluorescence microscope. Total RNA was extracted from THP-1 cells and THP-1 cells stably expressing ROP16 using the TRIzol reagent, followed by transcriptome sequencing. Differentially expressed genes (DEG) were screened, and a volcano plot was generated. DEGs were subjected to cluster analysis, Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway enrichment analysis, and gene ontology (GO) functional enrichment classification to screen immune response-related driver genes in human monocytic THP-1 cells following T. gondii infections, and gene expression was quantified using qPCR assay. Differences of means between groups were tested for statistical significance with independent sample t-test. Results Fluorescence microscopy displayed green fluorescence in more than 90% of the cells in the overexpression group in each field of view, and no green fluorescence was observed in the control group. qPCR assay quantified a higher relative expression level of ROP16 mRNA in the overexpression group (1 083.484 ± 68.990) than in the control group (1.000 ± 0) (t = 22.9, P < 0.01), indicating the successful generation of THP-1 cells that stably expressed ROP16. A total of 312 DEGs were identified in THP-1 cells stably expressing ROP16, including 193 upregulated genes and 119 downregulated genes. KEGG annotations showed that the highest proportion of DEGs were annotated to the organism system (24.2%), with 85 items significantly enriched, among which 47 genes were significantly enriched in the immune system. KEGG enrichment analysis showed that DEGs were significantly enriched in 20 signaling pathways, and 6, 7, and 8 DEG were significantly enriched in three pathways related to the immune system, including Th1 and Th2 cell differentiation, natural killer cell-mediated cytotoxicity, and IL-17 signaling pathways, respectively. GO functional annotations showed that a total of 309 DEGs were annotated to 55 secondary node classifications under three primary node classifications of biological processes, cellular components, and molecular functions. GO enrichment analysis showed that DEGs were significantly enriched in inflammatory response, negative regulation of tumor necrosis factor products, cytokine production, positive regulation of interferon-γ production, and immune response-related pathways. RT-qPCR assay detected that higher relative expression of MAN2B1, FOS, C1QA mRNA (25.994 ± 0.382、60.584 ± 2.968、 36.759 ± 0.180) in cells in the ROP16 overexpression group than in the control group (1.000 ± 0.039、1.000 ± 0.015、1.000 ± 0) (t = 92.00, 28.39, 280.7, P < 0.05 or 0.01), and lower relative expression of LMNB1, IL-6, and IL-12 mRNA (0.728 ± 0.054, 0.517 ± 0.073, 0.587 ± 0.015) in the overexpression group than in the control group (1.052 ± 0.027、1.000 ± 0.039、1.000 ± 0.010) (t = 7.64, 8.24, 33.62, P < 0.05 or 0.01). Conclusion The gene expression profile of human monocyte THP-1 cells changes significantly and may play important roles in the immune response following T. gondii infection.

Objective To generate the Toxoplasma gondii sub3 (Tgsub3) gene knockout strain using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, investigate the in-vitro phenotypes of the Tgsub3 gene knockout strain, and examine the effect of Tgsub3 gene on adhesion, invasion and proliferation of T. gondii. Methods SgUPRT on the pSAG1::CAS9U6::SgUPRT plasmid was mutated to single guide RNA (sgRNA) using site mutation, and the pSAG1::CAS9-U6::sgSUB3 plasmid with the sub3 gene knockout was generated. The DHFR resistant donor fragments containing 40 bp upstream and downstream homology arms of the sub3 gene were amplified, and the sub3 gene knockout plasmid and donor fragments were co-transfected into T. gondii by electroporation. Following resistance selection by pyrimethamine and monoclonal screening, the sub3 gene knockout strain RH∆ku80∆sub3 (∆sub3) was identified using PCR assay. The in-vitro phenotypes of the ∆sub3 strain were analyzed with plaque, invasion, and proliferation assays. Using RH∆ku80 strain as a control, all statistical analyses were conducted using the software GraphPad Prism 9. Results PCR assay identified bands with expected sizes in the ∆sub3 strain, indicating successful generation of the ∆sub3 strain. Plaque assay showed that the sizes of plaques formed by RH∆ku80 and ∆sub3 strains were (60.42 ± 23.20) au and (2.21 ± 1.89) arbitrary unit, respectively (t = 17.79, P < 0.01), and invasion assay showed that the invasion efficiencies of RH∆ku80 and ∆sub3 strains were (37.94 ± 18.18) % and (22.97 ± 15.36) %, respectively (t = 0.89, P > 0.05). Proliferation assay showed that the proportions of parasitophorous vacuoles containing 8 and more tachyzoites of RH∆ku80 and ∆sub3 strainswere (56.33 ± 8.58) % and (39.67 ± 11.84) %, respectively, and the proportions of parasitophorous vacuoles containing 4 and fewer tachyzoites of RH∆ku80 and ∆sub3 strains were (43.67 ± 8.58) % and (60.33 ± 11.84) %, respectively. Compared with control strain, the number of tachyzoites within the parasitophorous vacuole was significantly decreased (F = 17.93, P < 0.01). Conclusion The ∆sub3 gene knockout strain is successfully generatedand absence of the Tgsub3 gene affects the growth and reproduction of T. gondii tachyzoites.

Objective To investigate the inhibitory effect of andrographolide (AG) on Toxoplasma gondii and unravel its potential therapeutic mechanisms. Methods Human foreskin fibroblasts (HFFs) were treated with 0, 5, 10, 20, 40, 80, 120, 160, or 320 μmol/L AG and 10 μl CCK-8 solutions. The absorbance (A₄₅₀ value) was measured in HFFs following treatment and cell proliferation curves were plotted. Screening for concentrations that were not significantly toxic to cells for subsequent studies. For invasion assays, HFFs were divided into dimethyl sulfoxide (DMSO) group, AG group, and pyrimethamine (PYR) group, which were incubated in SAG1 antibody, green fluorescence-labeled goat anti-rabbit IgG antibody (1:1 000), and red fluorescence-labeled goat anti-rabbit IgG antibody (1:1 000), respectively; and then, T. gondii invasion was observed under an optical microscope, and the invasive rate was calculated. For proliferation assays, HFFs were divided into DMSO and AG groups, treated with green fluorescence-labeled goat anti-mouse IgG antibody (1:1 000), and T. gondii proliferation was observed under an optical microscope. For plaque assays, HFFs were divided into DMSO and AG groups, which were treated with 100 μl DMSO and 40 μmol/L AG, respectively, and the size of plaques were observed under an optical microscope. Interacting proteins were screened using surface plasmon resonance imaging (SPRi), and the interaction of isopentenyl diphosphate isomerase (IspG) gene with AG was validated with drug affinity responsive target stability assays. The IspG protein expression was determined using Western blotting assay, and the relative mRNA levels of interacting protein-coding genes were quantified using real time quantitative reverse transcription PCR (qPCR) assay. All statistical analyses were performed using the software GraphPad Prism 8.0.2. Results The results of proliferation experiments showed that the relative viability of HFF cells in 0, 5, 10, 20, and 40 μmol/L AG was 100.00%, 107.45%, 100.66%, 109.21%, and 90.94%, respectively, and the cell viability was maintained at the same level without obvious toxicity, and the relative viabilities of HFFs were 57.83%, 34.16%, 48.25%, and 30.75% following treatment with AG at doses of 80, 120, 160 and 320 μmol/L, appearing a remarkable toxicity (F = 14.96, P < 0.01). Indirect immunofluorescence assay revealed lower invasion rates in AG [(8.06 ± 2.40) %] and PYR groups [(6.36 ± 1.79) %] than in the DMSO group [(42.49 ± 9.75) %] (F = 35.88, P < 0.01), and the number of T. gondii parasitophorous vacuoles was higher in the DMSO group [(5.78 ± 0.94) parasites/vacuole] than in the AG group [(1.40 ± 0.12) parasites/vacuole] (t = 7.98, P < 0.01). Plaque assays showed a higher plaque size in the AG group (0 μm²) than in the DMSO group [(3 210 ± 1 840) μm²] (t = 19.03, P < 0.01). SPRi identified that the AG-interacting T. gondii proteins with the highest mass spectrometry scores included ribosomal RNA processing protein (2.19), ATP synthase α subunit (4.01), IspG (4.01), and START domain protein (2.12), appearing high molecular relevance. Drug affinity responsive target stability assays showed higher IspG expression in HFFs treated with 10 μmol/L AG (0.25 ± 0.01) than in controls (0.12 ± 0.01) (F = 294.2, P < 0.01). qPCR assay quantified higher relative IspG mRNA expression (4.903 ± 1.546) in HFFs treated with 40 μmol/L AG than in those treated with DMSO (1.19 ± 0.20) (t = 4.123, P < 0.05), and Western blotting determined that the IspG protein expression appeared a tendency towards a concentration-dependent manner, with relative IspG protein expression of 0.57 ± 0.01, 0.52 ± 0.02, 0.24 ± 0.05, and 0.03 ± 0.01 in HFFs treated with 0 (DMSO), 10, 20, and 40 μmol/L AG, respectively (F = 313.4, P < 0.01). Conclusion AG inhibits T. gondii invasion and proliferation through targeting IspG, appearing a remarkable anti-T. gondii activity.

Objective To investigate the prevalence of tick-borne piroplasm infections and the genetic evolutionary characteristics in equines in selected areas of Qinghai Province, so as to provide insights into management of equine piroplasmosis in Qinghai Province. Methods The blood samples were collected from free range equines in four cities (prefectures) of Haidong, Xining, Haixi and Haibei in Qinghai Province from March 2023 to July 2024. Genomic DNA was extracted from blood samples and the 18S rRNA gene was amplified using nested PCR assay. The obtained gene sequence was subjected to homology alignment and analysis using the NCBI BLAST program. Haplotype analysis was performed using the software DNASP 6, and a haplotype network diagram was plotted using PopArt. The sequences of Theileria equi genotypes A to E and Babesia caballi genotypes A to C were selected from GenBank as reference sequences, and a phylogenetic tree was built using the maximum likelihood estimation with the software MEGA 6.0. Results The prevalence of piroplasm infections infection was 47.73% (84/176) in 176 blood samples from free-ranging equines, and the prevalence rates of T. equi and B. caballi infections were 47.16% (83/176) and 0.57% (1/176), respectively. A total of 16 haplotypes were identified in the 83 sequences of T. equi, with a haplotype diversity of 0.450 and a nucleotide diversity of 0.026 43, and H1 was the dominant haplotype of T. equi (73.49%, 61/83), followed by H5 haplotype (9.64%, 8/83), and the remaining 14 haplotypes (H2 to H4, H6 to H16) each accounted for 1.20% (1/83). Phylogenetic analysis showed that the gene sequences of T. equi haplotypes H5, H8 to H14, H16 were clustered together with T. equi genotype A (GenBank accession numbers: JX177672 and MT463613) in GenBank into the same clade, and the remaining 7 haplotypes were clustered together with T. equi genotype B (GenBank accession numbers: EU642507 and AB515310) into the same clade, while the gene sequences of B. caballi and B. caballi genotype A (GenBank accession numbers: EU642512, KJ787774, AY309995, AB734392, MH651219, MH651220 and JX049130) in GenBank were clustered into the same clade. Conclusion T. equi and B. caballi are piroplasm species infecting equines in selected areas of Qinghai Province, with a high prevalence rate of infections. The prevalent genotypes are T. equi genotypes A and B and B. caballi genotype A, with T. equi genotype B as the dominant genotype.

Objective To analyze the epidemiological characteristics of Trichomonas vaginalis positive among women in the clinical laboratory of maternal and child health hospitals of Huzhou City, so as to provide a scientific basis for trichomoniasis control. Methods Data pertaining to T. vaginalis positive were retrieved from laboratory management information systems in Department of Laboratory Medicine of Huzhou Municipal Maternal and Child Health Hospital, Changxing County Maternal and Child Health Hospital, Anji County Maternal and Child Health Hospital, and Deqing County Maternal and Child Health Hospital from 2016 to 2023, including clinical manifestations, laboratory tests, treatments, and follow-up outcomes. Vaginal secretions were collected using sterile cotton swabs from the posterior fornix or cervical os. T. vaginalis positive were detected with a fully automated vaginal microecology analyzer among general cases, and using PCR assay among refractory or recurrent cases. Statistical analysis was performed using the software SPSS 26.0, and the effects of year, season, age, department and household registration on T. vaginalis positive rate were evaluated with analysis of Chi-square test. Results Among 875 407 vaginal secretion samples tested across four hospitals in Huzhou City from 2016 to 2023, 7 282 samples were positive for T. vaginalis positive. The highest positive rate was seen in 2016 (1.11%, 1 420/128 282), with the lowest in 2023 (0.45%, 419/93 517), and the positive rate appeared a tendency towards a decline over years from 2016 to 2023 (χ² = 499.746, P < 0.01), except for a minor rebound in 2021 (0.74%, 736/99 159). The highest positive rate was found in autumn (0.86%, 1 888/220 624), followed by in spring (0.83%, 1 859/225 219), winter (0.82%, 1 384/167 811) and summer (0.78%, 2 051/261 753), and the positive rate were 0.78% (192/24 740) in spring, 0.59% (188/31 786) in summer, 0.61% (157/25 931) in autumn and 0.80% (153/19 174) in winter in 2020 (χ² = 13.174, P < 0.05), while there were no season-specific positive rate in other seven years (χ² = 3.263, P > 0.05). The reported trichomoniasis cases had a median age of 39 years (range, 12 to 73 years), and the highest positive rate was seen among women at ages of 20 years and lower (1.57%, 405/25 780), followed at ages of 41 to 50 years (1.53%, 2 498/163 726), with the lowest among women at ages of 21 to 30 years (0.49%, 1 612/327 709) (χ² = 1 682.086, P < 0.01). There was a significant difference in the positive rate among women with (0.70%, 3 539/503 684) and without local household registration (0.92%, 3 581/371 723) (χ² = 180.228, P < 0.01), and the highest positive rate was seen in Department of Gynecology (1.10%, 6 129/557 907), followed by in Department of Physical Examinations (0.48%, 899/186 616) and Department of Obstetrics (0.24%, 308/130 884) (χ² = 1 312.963, P < 0.01). Conclusion The positive rate appeared a tendency towards a decline in Huzhou City over years from 2016 to 2023.

Objective To develop and establish an artificial intelligence (AI) platform assisting malaria parasites detection, and the applied effect was evaluated. Methods Blood smears were collected from current cases of overseas imported malaria in Wuhan City from 2008 to 2022. Microscopic images were captured with an automatic imaging collection system, and annotated by professional microscopists to create a dataset of thin blood smear images. A detection model was employed to detect Plasmodium, white blood cells (WBCs) and red blood cells (RBCs) to deduce negativity, positivity, infection rate and infected parasite species, and the results were displayed in the application interface. Based on numbers of true positive, true negative, false positive and false negative samples, the accuracy, precision, recall, and F1 score were estimated to evaluate the performance of the model. Microscopic examination procedures were mimicked according to the technical assessment method of malaria microscopists, and 10 blood smears were randomly selected to score the model comprehensive results. Results The thin blood smears image datasets included 3 704 images that were annotated by microscopist, including 7 835 Plasmodium annotations and 786 WBC annotations. Results from the YOLOv8 confusion matrix showed an overall accuracy of 85.5%, precision of 98.5%, recall of 93.3%, F1 score of 95.8%, and an mAP@0.50 score of 96.2%. The model had a high accuracy for detection of P. vivax (96.9%) and P. malariae (93.8%), and a low accuracy for detection of P. falciparum (87.2%) and WBCs (87.8%). Except an 89.7% F1 score for detection of P. falciparum, the model had F1 scores of over 90.0% for detection of other Plasmodium species and WBCs. The recall of the model for detection of 4 Plasmodium species and WBCs, was the highest recall for detection of P. malariae (94.9%), followed by WBC (94.2%), P. vivax (94.1%), P. ovale (93.6%) and P. falciparum (92.4%). A total of 64 targets were falsely identified, with a false detection rate of 6.7%. The correction rates of the platform and professional microscopist were both 100%, and the clinical microscopy score was 90 points, with no false positives or negatives identified; however, there were false detections in identification of malaria parasite species. Conclusion The platform is effective to identify and locate malaria parasites, WBCs and RBCs, and its scoring results are approaching to the microscopist. Due to a high detection capability, the model is feasible for auxiliary diagnosis of clinical microscopy of malaria parasites.

Objective To analyze the implementation of and evaluate the effectiveness of the schistosomiasis control strategy in Jiangxi Province during the stage of transmission interruption from 2016 to 2023, so as to provide insights into achieving the goal of schistosomiasis elimination. Methods The integrated strategy with an emphasis on management of the source of Schistosoma japonicum infection was implemented in Jiangxi Province from 2016 to 2023. Schistosomiasis control data were collected from 39 endemic counties (cities, districts) in Jiangxi Province, including examinations and chemotherapy for humans and livestock, snail control, health education, and multi-sectorial integrated control projects, and schistosomiasis surveillance data were extracted from the Information Management System for Parasitic Disease Prevention and Control. All statistical analyses were performed using the software SPSS 25.0, and the implementation of snail surveys and control, examinations and chemotherapy for schistosomiasis, disease surveillance, and integrated control measures was descriptively analyzed. Differences of proportions were tested for statistical significance with chi-square test. Results A total of 116 million Chinese Yuan were allocated to the schistosomiasis control programme in Jiangxi Province from 2016 to 2023, where the demonstration and promotion areas for the integrated strategy with an emphasis on management of the source of S. japonicum infection were built in 17 key schistosomiasis-endemic counties (cities/districts). As of the end of 2023, among 39 endemic counties (cities, districts), 24 achieved the criteria of schistosomiasis elimination and 15 achieved transmission interruption. Serological tests were performed for S. japonicum infections among 4 570 976 person-times in Jiangxi Province from 2016 to 2023, with annual seroprevalence of S. japonicum infections ranging from 1.68% to 3.21% (χ² = 4 833.74, P < 0.01), and the seroprevalence of S. japonicum infections appeared a tendency towards a decline among local residents (χ² = 360.88, P < 0.05) and floating populations (χ² = 156.82, P < 0.05) over years, with reductions of 47.88% and 47.58%, respectively. Parasitological tests were performed for schistosomiasis among 333 270 person-times, with no egg-positives detected since 2020. The number of current schistosomiasis cases decreased from 12 212 in 2016 to 5 454 in 2023 in Jiangxi Province, with a reduction of 55.34%, and the current schistosomiasis cases were all advanced cases since 2021, appearing a tendency towards a slow decline. The number of fenced livestock decreased from 101 630 in 2016 to 64 695 in 2023 in Jiangxi Province, with a reduction of 36.34%, with no egg-positive detected in bovines since 2020; however, there were two sites identified with S. japonicum infected wild stool samples in 2021 and one site with S. japonicum infected wild stool samples in 2023, including two hare stool samples and one bovine stool sample. No S. japonicum infection was detected in snails during the 8-year period from 2016 to 2023; however, the actual area of snail habitats increased by 8.69%, with an 8.48% increase in the area of snail habitats in marshland and lake regions and a 15.35% increase in hilly and marshland regions, respectively. The area of emerging snail habitats was 25.38 hm² (1 hm² = 10 000 m²), which was mainly detected in 2020 (0.89 hm²), 2021 (11.69 hm²), and 2022 (12.8 hm²). Results from schistosomiasis surveillance showed an 88.00% decline in the average density of living snails in national surveillance sites in Jiangxi Province. Conclusion The prevalence of schistosomiasis is low and appeared a tendency towards a decline in Jiangxi Province. However, a high priority requires to be given to control of emerging and re-emerging snails, wildlife surveillance, and management of cross-regional population mobility.

Objective To investigate the prevalence of soil-transmitted nematode infections in national surveillance sites of Guizhou Province from 2020 to 2023, so as to provide insights into formulation of the control strategy in the province. Methods National soil-transmitted nematodiasis surveillance sites were assigned in 9 cities (prefectures) of Guizhou Province from 2020 to 2023, including 6 fixed surveillance sites and 7 to 11 mobile surveillance sites each year. At each surveillance site, with the county as the unit, one administrative village was randomly selected from each of the five regions (eastern, western, southern, northern, and central) within the county to conduct monitoring. At least 200 permanent residents at ages of 3 years and older were randomly sampled from each administrative village, and at least 1 000 residents were randomly sampled from each surveillance site. Participants’ fecal samples were collected for identification of nematode eggs with the modified Kato-Katz thick smear method (two slides for each stool sample), and the time-, region- and population-specific prevalence rates of soil-transmitted nematode human infections were compared. Differences of proportions were tested for statistical significance with chi-square test. Results A total of 60 573 residents received surveillance on soil-transmitted nematode infections in Guizhou Province from 2020 to 2023, and the average prevalence of soil-transmitted nematode infections was 1.07% (651/60 573). The prevalence of soil-transmitted nematode infections was 1.81% (255/14 075), 1.32% (226/17 138), 0.75% (98/13 100), and 0.44% (72/16 260) from 2020 to 2023, respectively, which appeared a tendency towards a decline over years, and there was a significant difference among years (χ² = 155.7, P < 0.01). The average prevalence rates of Ascaris lumbricoides, hookworm, Trichuris trichiura infections were 0.43% (260/60 573), 0.37% (225/60 573) and 0.35% (213/60 573), respectively. The prevalence of both A. lumbricoides (0.89%, 0.50%, 0.24%, 0.11%) and T. trichiura infection (0.72%, 0.48%, 0.19%, 0.02%) appeared a tendency towards a decline over years from 2020 to 2023, and there were significant differences among years （χ² = 120.2, 123.1, both P < 0.01). The highest prevalence of soil-transmitted nematode infections was detected in Qiandongnan Prefecture (2.49%, 254/10 199), and the lowest in Guiyang City (0.10%, 7/7 036), showing a region-specific prevalence rate of infections (χ² = 416.4, P < 0.01). The prevalence of soil-transmitted nematode infections was 0.89% (262/29 396) and (1.25%, 389/31 177) among men and women, respectively. The highest prevalence was seen among residents at ages of 70 years and older 1.56% (106/6 808), followed by at ages of 3 to 9 years (1.46%, 132/9 012). The highest prevalence of soil-transmitted nematode infections was detected among Miao minority ethnic residents (2.09%, 160/7 639). The highest prevalence of soil-transmitted nematode infections was detected among students (1.44%, 188/13 019). In addition, the highest prevalence of soil-transmitted nematode infections was detected among illiterate residents (2.04%, 109/5 332). There were statistically significant differences among populations with different genders, age groups, ethnicities, occupations, and educational levels (χ² = 18.1, 92.7, 151.1, 56.6, 146.5，all P < 0.01). Conclusion The prevalence of soil-transmitted nematode infections appeared a tendency towards a decline in Guizhou Province over years from 2020 to 2023. However, higher attention should still be paid to key populations, including women, children, the elderly, ethnic minority residents, farmers (herdsmen), and individuals with low educational levels to facilitate the transmission control and interruption of soil-transmitted nematodiasis in Guizhou Province.

Objective To compare the differential expression of exosomal microRNAs (miRNAs) among alveolar echinococcosis (AE) patients pre- and post- surgery, and between AE and cystic echinococcosis (CE) patients, so as to investigate the value of exosomal miRNAs for diagnosis of echinococcosis. Methods Whole blood samples were collected from pre-surgery and 3 to 4 weeks post-surgery of 3 patients with clinically definitively diagnosed CE and 4 patients with clinically definitively diagnosed AE, 3 healthy individuals, and from 48 patients with typical AE lesions, 10 patients with typical CE lesions (all lesions detected by B-mode ultrasound), and 39 individuals negative for B-mode ultrasound. Whole blood samples were centrifuged and serum samples were collected. Exosomes were isolation and enriched using size exclusion chromatography and ultrafiltration, and RNA was extracted from exosomes for sequencing. MiRNAs were identified using miRDeep2, and miRNAs expression was quantified. The differential expression of exosomal miRNAs from patients was compared pre- and post-surgery using the edgeR software. Molecular probes were designed, and Echinococcus-derived exosomal miRNAs with high abundance were screened with qPCR assay and evaluated for their values in diagnosis of AE. In addition, the value of humans-derived differentially expressed miRNAs in differential diagnosis of echinococcosis was assessed. The comparison of relative expression levels of miRNA in human derived extracellular vesicles was performed using t-test. Results Exosomes appeared circular or circular-like vesicular structures of varying sizes and uneven distribution under a transmission electron microscope, with diameters ranging from 30 to 150 nm and distinct membranous structures. A total of 83 Echinococcus-derived miRNAs were identified in the exosomes from AE patients’ pre-surgical serum samples, including 64 known miRNAs and 19 novel miRNAs, and most miRNAs had low expression in serum samples, with emu-let-7-5p showing relatively high expression. Two miRNAs with high abundance (emu-miR-71-5p and emu-miR-10-5p) screened by qPCR assay and emu-let-7-5p that had been reported to exhibit a diagnostic value were included for further verification. The detection of emu-let-7-5p was 20.83% (10/48)；emu-miR-71-5p was detected in both positive and negative samples, with detection rates of 87.50% (42/48) and 30.76% (12/39), respectively; while emu-miR-10-5p was only detected in 52.08% (25/48) of positive samples. A total of 8 differentially expressed humans-derived exosomal miRNAs were identified, and 5 were selected for evaluation based on their expression abundance. Among them, 3 miRNAs (has-miR-183-5p, has-miR-222-3p and has-miR-196a-5p) had average expression levels of 355, 299, and 213 in CE positive serum samples, which were 39.5, 10.0, and 23.6 times higher than negative controls, respectively, and the difference was statistically significant (t = 7.15, 5.45, 6.26, all P < 0.01). Conclusion Due to trace levels in serum, Echinococcus-derived exosomal miRNAs still have limitations as diagnostic markers for echinococcosis. Three humans-derived exosomal miRNAs have potential in CE diagnosis.

Objective To investigate the species and microbial diversity of ticks from three autonomous prefectures in Guizhou Province. Methods In April 2019 and July 2020, parasitic ticks on the body surfaces of cattle, sheep and rodents were collected in Qiandongnan Miao and Dong Autonomous Prefecture (referred to as Qiandongnan), Qiannan Buyi and Miao Autonomous Prefecture (referred to as Qiannan) and Qianxinan Buyi and Miao Autonomous Prefecture (referred to as Qianxinan) in Guizhou Province, followed by morphological identification. The ticks were divided into 6 groups based on the collection regions and tick species. After DNA extraction, 16S rDNA high-throughput sequencing was performed. The sequencing results were analyzed by operational taxonomic unit (OTU) classification cluster and compared with the ribosomal database to obtain species annotations, enabling bacterial community composition analysis and α diversity analysis. Hierarchical clustering was conducted based on the β diversity distance matrix and a sample clustering tree was constructed using the Bray-Curtis algorithm. Non-metric multidimensional scale (NMDS) analysis and inter-group similarity analysis were performed using R 3.6.3 software. Results A total of 1 463 ticks were collected, including 1 227 Rhipicephalus microplus (83.87%), 208 Haemaphysalis longicornis (14.22%), 25 Ixodes granulatus (1.71%) and 3 Haemaphysalis flava (0.20%). In Qiandongnan, Qiannan and Qianxinan, 220, 1 039 and 204 ticks were collected respectively, accounting for 15.04%, 71.02% and 13.94%. A total of 1 682 OTUs were generated. α diversity analysis revealed that H. longicornis from Qiandongnan exhibited relatively high Shannon index, Chao1 index, Ace index and evenness index values of 4.868, 568.481, 567.479 and 0.770, respectively. Bacterial community identification annotated a total of 29 phyla, 70 classes, 118 orders, 245 families and 503 genera. Proteobacteria, Alphaproteobacteria, Rickettsiales, Rickettsiaceae and Rickettsia emerged as the dominant taxa at their respective taxonomic levels, with average relative abundances of 75.52%, 57.70%, 52.47%, 50.88% and 50.88%, respectively. The bacterial genera carried by R. microplus were predominantly Rickettsia (89.45%) and Coxiella (2.82%), while H. longicornis mainly harbored Rickettsia (18.06%) and Pseudomonas (11.98%), and I. granulatus primarily carried Spiroplasma (33.19%) and Staphylococcus (22.22%). The dominant bacterial genus in ticks from Qiandongnan, Qiannan, and Qianxinan was Rickettsia, with average relative abundances of 46.96%, 60.20% and 45.47%, respectively. The sample clustering tree demonstrated that R. microplus samples from 3 regions clustered together with a H. longicornis sample from Qiannan, while the remaining H. longicornis samples formed a separate cluster, and 3 I. granulatus samples from Qianxinan clustered independently. NMDS and inter-group similarity analysis indicated distinct bacterial community compositions among the 6 sample groups (R = 0.599, P < 0.01). Conclusion The bacterial communities carried by ticks in the three autonomous prefectures of Guizhou Province exhibited rich diversity, with Rickettsia being the dominant genus. Additionally, the bacterial community compositions differed among various tick species.

Objective To decipher the mitochondrial genome structure and phylogenetic relationships of Rhipicephalus turanicus and investigate the genetic differentiation among different geographical populations of R. turanicus, so as to provide data supports to species identification and phylogenetic analysis. Methods A male R. turanicus tick was collected and identified from the body surface of a domestic dog in Mapo Township, Weishan County, Jining City, Shandong Province on August 2023, and genomic DNA was extracted from the tick for second-generation sequencing on an Illumina high-throughput platform. The complete mitochondrial genome of R. turanicus was assembled using Novoplasty software and the functions of the complete mitochondrial genome were preliminarily annotated with the MITOS software. In addition, phylogenetic trees were constructed using the maximum likelihood method based on the sequences of 13 protein-coding genes (PCGs). Results Second-generation sequencing showed that the complete mitochondrial genome of R. turanicus was 14 718 bp in length (GenBank accession No.: PV416795), which was consisted of 13 PCGs, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 2 non-coding regions (NCRs). The complete mitochondrial genome contained overlapping regions or intergenic regions, and the base composition exhibited a clear A + T preference. The PCGs initiated with four typical start codons ATN (ATA, ATC, ATG, and ATT), and terminated with stop codons TAA, TAG, TA and T. Among the secondary structures of 22 tRNA gene, all formed a typical cloverleaf structure except for trnC, trnF, and trnS1, which lacked the dihydrouracil (DHU) arm and DHU loop, resulting in failure in formation of a complete cloverleaf structure. Phylogenetic analysis revealed that the R. turanicus sampled from Shandong Province in this study was clustered with tick populations from Beijing (GenBank accession No.: OM368330) and Xinjiang (GenBank accession No.: KY996841, NC035946, OM368326) into the same clade branch, and was grouped into a strongly supported monophyletic clade with populations from Israel (GenBank accession No.: OQ184023) and Saudi Arabia (GenBank accession No.: PP919886). The R. turanicus populations from different geographic regions were clustered into a highly supported phylogenetic clade, suggesting a high degree of genetic conservation and a tendency towards regional genetic differentiation. Conclusion The complete mitochondrial genome sequence of R. turanicus from Shandong Province has been obtained, which conforms to the mitochondrial genome characteristics of the Ixodidae family, and there is a tendency towards potential genetic differentiation among different geographic populations of R. turanicus.

Objective To retrospectively analyze the species composition, list of classification, and infection prevalence of chigger mites in Liangshan Yi Autonomous Prefecture, Sichuan Province, so as to provide insights into monitoring and prevention and control of scrub typhus and its vector chigger mites in the prefecture. Methods Field surveys were conducted in six counties of Muli Tibetan Autonomous County, Mianning County, Zhaojue County, Yanyuan County, Dechang County, and Huidong County in Liangshan Yi Autonomous Prefecture from 2013 to 2020. Chigger mites were collected from the surface of murines and small mammals in different habitats, enveloped with Hoyer’s medium to generate slide specimens of chigger mites, and individually characterized to species. The prevalence (P_M), mean abundance (MA), and intensity of chigger mite infections (MI) were calculated, and the community indicators of chigger mites were estimated, including richness index (S), Shannon Wiener diversity index (H’), Pielou evenness (E), and Simpson dominance index (D). The adequacy of sampling was determined using species rarefaction curves, extrapolation curves, and sample coverage. In addition, the interspecific relationships among main chigger mites were examined using Spearson correlation coefficients, and the interrelationships between host animals and chigger mites were evaluated using bilateral network analysis. Results A total of 20 313 chigger mites were identified from the surface of 778 murines and other small mammal hosts belonging to 24 species, 14 genera, 4 families and 3 orders, including 132 species (15 vector or potential vector chigger mites), 14 genera, 3 subfamilies and 2 families, and Leptotrombidium was the dominant genus (75.3%, 15 296/20 313). The gross P_M, MA, MI, S, H’, E and D of chigger mites were 65.2%, 26.11 mites/animal, 40.07 mites/animal, 32, 3.20, 0.65, and 0.92 on the surface of small mammals across Liangshan, with sample coverage of 99.9%. The P_M, MA and MI of chigger mites appeared a tendency towards a decline with the rise in elevation, and the community indicators of chigger mites appeared a tendency towards a rise followed by a decline with the rise in elevation. Of 132 species of chigger mites, the constituent ratio of five dominant species was 52.4%, and there were positive correlations with varying degrees among 5 dominant chigger mites and 5 main vector chigger mites (r = 0.19-0.87, P < 0.01). Bilateral network analysis revealed that one host harbored multiple species of chigger mites and one species of chigger mites was parasitized in multiple species of hosts. Conclusion Chigger mites are abundant in species and high in species diversity in Liangshan Yi Autonomous Prefecture. The prevalence of chigger mite infections is common and high in hosts, and the prevalence of chigger mite infections and community structure of chigger mites vary in elevation. The main chigger mites have a tendency towards choosing the same host and interspecific coexistence in Liangshan, and Leptotrombidium genus is widespread in Liangshan. There are 15 species of vector chigger mites, and most present a low specificity to hosts, which increases the potential risk of continuous transmission of scrub typhus in Liangshan.

Objective To investigate the composition, structure and function of the microbial community in Dermatophagoides farinae and to analyze characteristics of their functional genes. Methods Total DNA was extracted from D. farinae and paired-end library was constructed, and metagenomic next-generation sequencing (mNGS) was performed following bridging PCR assay. The obtained gene sequences aligned with BLAST program in the non-redundant protein sequence (NR), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Carbohydrate Activating Enzymes (CAZY) and the Comprehensive Antibiotic Resistance (CARD) databases. The diversity of microbial genes was analyzed in D. farinae with observed species index, Ace index, Chao1 index, Shannon diversity index, Simpson’s diversity index and coverage index. Results mNGS results of D. farinae yielded (1.18 to 1.29) × 10⁷ bases. Following sequence assembly, there were 34 527 overlapping groups, with an N50 value of 25 005, and the mean length of overlapping groups was 11 509. The top 20 most abundant species were annotated to 73 phyla, 430 genera, and 689 species, and bacteria was the predominant microorganism in D. farina (80.9%), followed viruses (5.5%) and fungi (1.2%) in D. farinae. Within the bacterial community, Proteobacteria (97.8%) and Firmicutes (0.8%) were dominant phyla, with Bartonella (59.0%) and unclassified Alphaproteobacteria (11.0%) as dominant genera. Within the fungal community, Microsporidia (66.6%) and Ascomycota (19.6%) were dominant phyla, with Pancytospora (41.4%) and Saccharomyces (12.2%) as dominant genera. In the viral community, Cressdnaviricota (96.2%) and Uroviricota (2.6%) were dominant phyla, with Orbivirus (96.2%) as the dominant genus. α diversity analysis showed a good repeatability of D. farinae samples. Protein sequence annotation results in the EggNOG database showed that bacteria and fungi were annotated to 23 and 22 functional categories, respectively, with the percentage of genes associated with translation/ribosome structure and biogenesis (bacteria, 15.3%; fungi, 27.4%). KEGG annotation results showed that cofactor- and vitamin metabolism-related genes were dominant in bacteria (10.5), and genes associated with folding, sorting, and degradation were more pronounced in fungi (12.0%). CAZY annotations showed that 98.3% of bacterial genes were matched to 65 distinct CAZY families, with glycosyltransferases as the dominant CAZY family member (57.6%), and only 0.4% of fungal genes were annotated to 12 CAZY families, with glycosyltransferases as the primary CAZY family member (87.7%). CARD annotations showed that 99.1% of bacterial genes were annotated to 17 diverse antibiotic resistance genes, with multidrug resistance genes exhibiting the highest prevalence (39.5%), and 0.3% of fungal genes were annotated to 5 antibiotic resistance genes, with tetracycline resistance genes (41.6%) and multidrug resistance genes (39.7%) as the two most common genes. Conclusion The microbial community within D. farinae exhibits a significant complexity and diversity, and the gene functions and metabolic pathways vary between bacteria and fungi.

Objective To develop a molecular assay for identification of common Culicoides species in Fujian Province based on nested PCR assay, and to investigate the genetic evolution of Culicoides. Methods Culicoides biting midges were captured using light traps in Fujian Province from June 2023 to July 2024 and mounted on microscope slides for morphological identification. The specific primers for nested PCR assay targeting the mitochondrial cytochrome C oxidase subunit 1 (cox1) sequence of Culicoides were designed and synthesized. Genomic DNA was extracted from Culicoides for nested PCR assay and conventional PCR assay, and the amplification products were checked with agarose gel electrophoresis and then compared with morphological dentification results. Following nested PCR assay, the amplification products were sequenced and the resulting reads were assembled using the SeqMan Pro software. The nucleotide homology searches were conducted using the BLAST tool, and the cox1 sequences were aligned using the Clustal X software. The haplotype network was constructed and the haplotype diversity (Hd) index was calculated using the WinArl35 and popart software. The sample sequences were characterized using the MEGA 11 software, and a phylogenetic tree was built using the neighbor-joining method. The intra-population and inter-population genetic distances among different Culicoides species were calculated with P-distance model. Results A total of 35 biting midge samples were collected and morphologically identified as 12 species, including C. arakawai, C. homotomus, C. oxystoma, C. huffi, C. morisitai, C. sumatrae, C. maculatus, C. clavipalpis, C. circumbasalis, C. orientails, Leptoconops chinensis and Lasiohelea wuyiensis. Nested PCR assay successfully amplified specific bands for 29 DNA samples from morphologically identified Culicoides biting midges, with a 100% detection rate of Culicoides (29/29), and conventional PCR assay amplified 17 specific bands with low brightness for 29 DNA samples from morphologically identified Culicoides biting midges, with a 58.6% detection rate of Culicoides (17/29). The BLAST results demonstrated that 4 cox1 sequences of morphologically identified C. circumbasalis had the highest homology (91.64%-91.78%) with the sequence of C. flumineus (ON002367) in Genbank, and had 84.78%-84.87% homology with the reference sequence of C. circumbasalis (KY441782), while 3 cox1 sequences of morphologically identified C. orientails showed the highest homology (91.64%-91.78%) with the sequence of C. flumineus (ON002367) and had 81.93%-82.42% homology with the reference sequence of C. orientails (KT352219). The BLSAT results of other 22 cox1 sequences were consistent with morphological identification results, with sequence homology ranging from 86.78%-100%. The haplotype network diagram of the cox1 gene revealed that the 29 cox1 sequences contained 22 haplotypes, with considerable genetic variation among different Culicoides species (Hd = 0.968 0). Phylogenetic analysis revealed that the 29 cox1 sequences were clustered into the respective clades according to their corresponding subgenera. The inter- and intra-population genetic distances were 0.000 4-0.222 9 and 0.000 0-0.006 2 among different Culicoides species. Conclusion The nested PCR assay-based molecular identification was built, and it could be effectively applied to multiple common Culicoides species in Fujian Province.

Objective To investigate the trends in disease burden and distribution characteristics of major human parasitic diseases in the world from 1990 to 2021, so as to provide insights into parasitic disease control. Methods Age-standardized incidence, prevalence, mortality, and disability-adjusted life years (DALYs) of 12 major human parasitic diseases, including malaria, American trypanosomiasis, leishmaniasis, African trypanosomiasis, schistosomiasis, cysticercosis, cystic echinococcosis, lymphatic filariasis, onchocerciasis, intestinal nematode infections, food-borne trematodiases, and guinea worm disease, were extracted from the Global Burden of Disease database, and the temporal, population (gender) and regional [including socioeconomic index (SDI) stratification] distributions of and trends in disease burdens were analyzed. R software was used for data processing and visualization, and Monte Carlo simulation was employed to estimate uncertainty intervals (UI). Results The total global DALYs of parasitic diseases declined from 74.03 million to 64.24 million person-years from 1990 to 2021, with a reduction of 13.22%. The disease burdens due to most types of parasitic diseases declined, and the disease burdens due to onchocerciasis, cysticercosis, food-borne trematodiases and American trypanosomiasis increased by 1 to 5 folds. As to types of parasitic diseases, malaria always bore the highest disease burden (consisting of more than 3/4 of total burdens), and the disease burden due to leishmaniasis fell from the 2nd to the 8th place, while the burden of schistosomiasis rose to the 2nd place. The disease burden of malaria had remarkably reduced since 2005 and rebounded slightly in recent years, and the burdens due to intestinal nematode infections and lymphatic filariasis have declined by more than 50%. The highest burden of parasitic diseases was found in sub-Saharan Africa, with high clustering of disease burdens due to malaria and schistosomiasis, and the disease burden due to food-borne trematodiases was concentrated in east Asia and Southeast Asia, while cysticercosis was also highly prevalent in Europe and the United States. The disease burden due to lymphatic filariasis was higher among men (the second leading cause of death) than women (the seventh leading cause of death); malaria and schistosomiasis ranked 1st and 3rd respectively among the causes of death for males, and 1st and 2nd respectively among the causes of death for females. The disease burden due to parasitic diseases was significantly higher in low SDI regions than in high SDI areas. Conclusion The overall global burden of parasitic diseases has declined overall; however, regional disparities are prominent, and the risk of rebound for some parasitic diseases remains. It is necessary to adjust the parasitic disease control strategies, strengthen resources investment in low SDI regions, pay attention to gender differences, and be alert to emerging challenges such as climate change and globalization.

Calreticulin is a soluble Ca²⁺-binding protein that is mainly present endoplasmic reticulum, which has a variety of biological functions, such as Ca²⁺ signal transduction, protein folding, and antigen presentation. The calreticulin of parasitic helminths is secreted by the worm body or expressed on the surface of the worm body, which plays an important role in the parasite motility, growth and development. In addition, the calreticulin of some helminths is strongly associated with its immune evasion process. This review summarizes the structure composition and function of calreticulin, and discusses the mechanisms underlying the regulation of host Th1/Th2 immune responses and the regulatory mechanisms of host immune cells, immune molecules and complement system by calreticulin, so as to provide new insights into management of helminthiasis.

Apicomplexan protozoa are a class of obligate intracellular parasites, and most of them are pathogens of zoonoses, with a wide range of hosts, causing huge economic losses to the livestock breeding industry and posing great threats to public health security. It has been demonstrated that protein disulfide isomerase (PDI) is widely present in apicomplexan protozoa, which is mainly associated with the invasion, adhesion and cellular immunity of parasites and plays an important role in the growth and development process of parasites. This review summarizes the advances in the structure and biological functions of PDI in apicomplexan protozoa, so as to provide insights into management of parasitic diseases and research and development of vaccines against parasitic diseases.

Toxoplasma gondii is a globally distributed obligate intracellular parasite that causes zoonotic toxoplasmosis. Studies have found that chronic T. gondii infection leads to damage of the host’s central nervous system (such as altered neuroimmune response and neurotransmitter imbalance) through the formation of tissue cysts in the host brain, manifested as changes in mental disorders (such as schizophrenia, bipolar affective disorder, major depression, and suicide attempts), cognitive decline, Alzheimer’s disease, and behavioral changes (such as fatal attraction phenomenon, increased risk of traffic accidents, increased probability of becoming a wolf king, increased sexual attractiveness, and changes in dietary preferences), etc. This review aims to explore the relationship and underlying mechanisms between chronic T. gondii infection and host neuropsychiatric and behavioral changes.

To investigate the prevalence of Paragonimus metacercariae infections and genotyping of Paragonimus metacercariae in freshwater crabs from Jianning County, Fujian Province, freshwater crabs were collected from mountain streams and ditches near residential areas along the Chuxi River system in Jianning County, Fujian Province, and Paragonimus metacercariae were collected by twice filtering through the sieves for morphological characterization. Genomic DNA was extracted from Paragonimus metacercariae, and the ribosomal internal transcribed spacer 2 (ITS2) gene sequence was amplified using PCR assay. Following sequencing of the PCR amplification product, the gene sequence was uploaded to the GenBank database, and gene sequence alignment was performed using the BLAST tool. A phylogenetic tree was built using the neighbor-joining method with the MEGA software with Fasciola hepatica as an outgroup. A total of 71 freshwater crabs were captured, and there were 60 crabs infected with Paragonimus metacercariae, with an 84.51% prevalence rate of infections. A total of 782 Paragonimus metacercariae were identified, and the intensity of infections was 13 metacercariae per crab. The PCR amplification product were approximately 500 bp in length. The ITS2 gene sequence from this study (GenBank accession No.: PQ510326.1) was 99.13%, 98.79%, and 99.18% identical to P. westermani isolates from Jilin Province (GenBank accession No.: AB713404.1), Shanghai Municipality (GenBank accession No.: KC417492.1), and Zhejiang Province (GenBank accession No.:JQ354935.1), respectively. Phylogenetic analysis revealed that the two gene sequences obtained from this study were clustered into the same clade with sequences of P. westermani isolates from Jilin Province (GenBank accession No.:AB713404.1), Shanghai Municipality (GenBank accession No.: KC417492.1), and Zhejiang Province (GenBank accession No.: JQ354935.1) at a genetic distance of 0, and they exhibited genetic distances of 0.079 and 0.075 P. skrjabini isolates (GenBank accessions No.: AB325516.1 and AB703444.1) and 0.078, and 0.078 from P. miyazakii isolates (GenBank accessions No.: AB713405.1 and AY618741.1), with distinct clade separation. The gene sequences of P. westermani isolates from this study share high identities to those of P. westermani isolates from other provinces, suggesting that these P. westermani isolates may serve as a reference isolate of P. westermani in Fujian Province.

To compare the immunogenicity of excretory/secretory products of Clonorchis sinensis (CsESP) at different intervals and evaluate their diagnostic value for clonorchiasis. Adult C. sinensis were cultured in vitro, and the culture supernatants collected 0-24 hours and 24-48 hours were mixed to prepare CsESP-1 and CsESP-2 antigens, respectively. Mice were randomly divided into 4 groups: CsESP-1, CsESP-2, adjuvant and PBS groups. Mice in each group were subcutaneously immunized with the corresponding antigen. Blood samples were collected at weeks 0, 2, 4 and 6 post-immunization to prepare immune sera. ELISA was used to measure the antibody titers against CsESP and levels of IgG and IgG1. Additionally, ELISA was employed to detect the levels of CsESP-specific IgG and its subclasses in blood samples from healthy individuals and those infected with C. sinensis. SPSS 27.0 software was used to calculate sensitivity, specificity and Youden’s index. The receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were computed to evaluate the diagnostic efficacy of the antigens. Blood samples from healthy individuals (Cs^-) and those infected with C. sinensis (Cs⁺), Strongyloides stercoralis, Fasciola gigantica and malaria patients were tested for cross-reactivity using ELISA. Comparisons between two groups were conducted using the t-test, while paired ROC analysis was used for AUC comparisons. ELISA results showed that at week 6 post-immunization, the antibody titers against CsESP in the sera of mice in the CsESP-1 and CsESP-2 groups were 1:102 400 and 1:400, respectively, indicating superior immunogenicity of CsESP-1. At week 6 post-immunization, the A₄₅₀ values for specific IgG and IgG1 in the sera of mice in the CsESP-1 group were 0.926 and 1.803, respectively, both significantly higher than those in the adjuvant group (0.147 and 0.080, respectively) (t = 5.805, 5.643; both P < 0.05). In the CsESP-2 group, the A₄₅₀ values for specific IgG at weeks 2, 4 and 6 post-immunization were 0.279, 0.437 and 0.523, respectively, all higher than those in the adjuvant group (0.133, 0.127, 0.142) (t = 2.906, 5.286, 6.83; P < 0.05, 0.05, 0.01). The A₄₅₀ values for IgG1 in the CsESP-2 group at weeks 4 and 6 post-immunization were 0.225 and 0.332, respectively, both higher than those in the adjuvant group (0.095, 0.083) (t = 3.423, 2.485; both P < 0.05). In Cs⁺ blood samples, the A₄₅₀ values for CsESP-1-specific IgG, IgG2 and IgG4 were 0.817, 0.274 and 0.524, respectively, all higher than those in Cs^- blood samples (0.456, 0.102 and 0.103, respectively) (t = 7.184, 2.720, 5.634; all P < 0.01). For CsESP-2, the A₄₅₀ values in Cs⁺ blood samples for specific IgG, IgG1, IgG2 and IgG4 were 0.783, 0.132, 0.233 and 0.465, respectively, all higher than those in Cs^- blood samples (0.460, 0.068, 0.074 and 0.082, respectively) (t = 4.768, 2.519, 2.634, 4.991; P < 0.01, 0.05, 0.05, 0.01). The CsESP-1 antigen demonstrated the best overall performance in detecting IgG4 in Cs⁺ blood samples, with sensitivity, specificity and accuracy of 89.2%, 86.5% and 87.9%, respectively. Similarly, the CsESP-2 antigen showed the same sensitivity specificity and accuracy (89.2%, 86.5% and 87.9%, respectively) for detecting IgG4 in Cs⁺ samples. ROC curve analysis revealed that the CsESP-1 antigen had moderate diagnostic value for detecting IgG1 and IgG2 (both AUC > 0.7) and high diagnostic value for detecting IgG and IgG4 (both AUC > 0.9). The CsESP-2 antigen exhibited moderate diagnostic value for detecting IgG, IgG1 and IgG2 (all AUC > 0.7) and high diagnostic value for detecting IgG4 (AUC > 0.9). Paired ROC analysis indicated that the diagnostic efficacy of the CsESP-1 antigen for IgG and IgG1 was superior to that of the CsESP-2 antigen (z = 3.515, 2.149; P < 0.01, 0.05). Neither the CsESP-1 nor CsESP-2 antigens showed cross-reactivity with IgG in blood samples from individuals infected with S. stercoralis, F. gigantica or malaria patients. In conclusion, both the CsESP-1 and CsESP-2 antigens prepared in this study exhibited good immunogenicity and could serve as candidate antigens for the diagnosis of clonorchiasis.

A 9-year-old Buyi boy, a student from Guizhou Province (a Paragonimus-endemic region), presented to the First Affiliated Hospital of Guizhou Medical University in June 2024 with sudden-onset right-sided limb weakness. His medical history was significant for chronic consumption of untreated water and raw crustaceans (shrimp and crabs). Initial cranial CT revealed left frontoparietal hemorrhage, while chest CT demonstrated a cavity in the left lower lung lobe. Laboratory tests showed marked eosinophilia (18.40%). Initially diagnosed as “intracerebral hemorrhage of unknown etiology”, he was treated with sodium valproate oral solution (20 ml/d) for 3 weeks, leading to symptomatic remission. In October, the patient was readmitted due to recurrent intracerebral hemorrhage with vomiting. Laboratory tests showed marked eosinophilia (18.10%). Enhanced brain MRI showed multiple ring-enhancing lesions in the left frontotemporoparietoccipital lobes, and chest CT revealed migratory pulmonary cavitations. Serological confirmation via peripheral blood ELISA showed positive Paragonimus IgG antibody, establishing the diagnosis of cerebro-thoracic paragonimiasis. Treatment consisted of praziquantel [75 mg/(kg·d) orally, divided into three doses daily for 3 days, repeated after a 1-week interval], dexamethasone tablets (0.75 mg/d orally), and continued sodium valproate (20 ml/d orally). At 1-month follow-up, imaging showed resolution of the intracerebral hemorrhage and reduction in pulmonary lesions, with seroconversion of Paragonimus antibody. Six months post-discharge, the child exhibited full recovery of right limb muscle strength without recurrent hemorrhage.

A 39-year-old female worker with her ancestral home in Shaanxi, had lived, studied, and worked in Golmud, Qinghai since birth until now. She was admitted to the Army 952nd Hospital of the People’s Liberation Army of China due to gallbladder stones on March 1, 2022. Ultrasound examinations showed multiple gallbladder stones, an enlarged gallbladder, and bile stasis, and CT scans displayed gallbladder stones. Routine blood tests showed a total white blood cell count of 9.59 × 10⁹/L, a neutrophil percentage of 70.8% (elevated), an eosinophil count of 0.01 × 10⁹/L (decreased), eosinophil percentage of 0.1% (decreased), alanine aminotransferase activity of 68.3 U/L (elevated), aspartate aminotransferase activity of 50 U/L (elevated), total protein of 59 g/L (decreased), and albumin of 37.7 g/L (decreased). Physical examination revealed tenderness in the upper-middle right abdomen. The patient underwent a laparoscopic cholecystectomy on March 2, and the pathological examination of the gallbladder identified Clonorchis sinensis eggs. The patient had no history of living in endemic areas and only had a habit of consuming sliced raw fish in Qinghai. Based on epidemiological history, clinical symptoms, and relevant auxiliary examination results, she was definitively diagnosed with C. sinensis infection, and was the first local case in Qinghai. The patient was cured and discharged from hospital on March 9, and was treated with praziquantel (at a total dose of 210 mg/kg, three times daily for successively 3 days). Subsequent multiple fecal examinations did not show any abnormalities, and no abnormalities were found at 3-month and 2-year follow-up visits.