Table of Content

30 April 2026, Volume 44 Issue 2

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SPECIAL REPORT

Epidemiological characteristics of malaria in China, 2025

ZHANG Li, XIA Zhigui, LI Shizhu

2026, 44(2):  153-157.  doi:10.12140/j.issn.1000-7423.2026.02.001

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Malaria case investigation forms in 31 provinces/municipalities/autonomous regions (excluding Taiwan, Hongkong, Macao) and the Xinjiang Production and Construction Corps aross China in 2025 were collected from the National Notifiable Infectious Diseases Reporting Information System, and the epidemiological characteristic of malaria cases were statistically analyzed. A total of 4 314 malaria cases were reported in China in 2025, which increased by 50.8%, 439.9%, and 36.6% relative to in 2017 (2 861), 2021 (799) and 2024 (3 157), respectively, and 11 deaths were reported in 2025, which reduced by four as compared to in 2024 (15 deaths). Of all reported malaria cases, there were 2 829 Plasmodium falciparum malaria cases (65.6%, 2 829/4 314), 963 P. vivax malaria cases (22.3%. 963/4 314), 372 P. ovale malaria cases (8.6%, 372/4 314), 103 P. malariae malaria cases (2.4%,103/4 314), and 47 cases with mixed infections (1.1%, 47/4 314), and there were 4 308 imported cases, 5 non-mosquito-borne malaria cases (transfusion-transmitted infection), and one recrudescent case with a long incubation period (P. malariae malaria). Malaria cases were reported in 30 provinces and the Xinjiang Production and Construction Corps, with the exception of Xizang Autonomous Region, with Yunnan Province (678 cases), Guangdong Province (359 cases), Henan Province (354 cases), Hunan Province (338 cases), and Shandong Province (307 cases) as the five most seriously affected provinces, accounting for 47.2% (2 036/4 314) of totally reported malaria cases. The ratio of male to female cases was 12.8 ∶ 1, and patients at ages of 30 to 39 years accounted for the largest proportion of cases (29.1%, 1 255/4 314). The most common reason for exit and entry was labor (71.6%, 3 086/4 308). A total of 131 critically ill malaria cases were reported across 27 provinces in 2025 (3.0%, 131/4 314), which increased by 17.0% relative to in 2024 (112 cases). Malaria epidemic has risen for four consecutive years in China, and thousands of imported malaria cases have posed substantial pressure and challenges to prevention of re-establishment of imported malaria.

ORIGINAL ARTICLES

Immunological characteristics of splenic dendritic cells subsets in mice at different stages of Echinococcus multilocularis infection

TANG Na, AYINAER Jiensi, XIAO Wenying, ABIDAN Ainiwaer, SUN Sheng, GE Conghui, WANG Mengying, GAO Yi, HU Qiu, LI Jing, ZHANG Chuanshan, WANG Hui

2026, 44(2):  158-165.  doi:10.12140/j.issn.1000-7423.2026.02.002

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Objective To investigate the changes in the number, proportion and immunological characteristics of splenic dendritic cells (DCs) and their subsets in mice at different stages of Echinococcus multilocularis infection. Methods C57BL/6J mice were randomly divided into a control group and an infection group. Mice in the infection group were injected with 4 000 E. multilocularis protoscoleces via the hepatic portal vein, while mice in the control group received an equal volume of normal saline. Spleen tissues were collected at 2 weeks and 24 weeks post-infection, respectively. Paraffin-embedded sections were subjected to immunohistochemistry (IHC) to observe the localization of CD11c⁺ cells in spleen. Splenic lymphocytes were isolated, and flow cytometry was used to detect the number, proportion and expression of co-stimulatory molecules CD40 and CD80 on DCs and their subsets in the spleen of both groups. Results IHC results showed that CD11c⁺ cells in both the control and infection groups were predominantly localized in the marginal zone of the splenic white pulp at all infection stages, with no statistically significant difference in the proportion of positive staining area between groups (t = 0.473, -1.885; both P > 0.05). Flow cytometry revealed that at 2 and 24 weeks post-infection, splenic DCs in the infection group accounted for (11.33 ± 2.67)% and (15.24 ± 3.63)% of total splenic lymphocytes, respectively, both significantly lower than those in the control group [(18.08 ± 3.47)% and (22.06 ± 4.58)%, respectively] (t = 3.449, 2.761; P < 0.01, 0.05). The proportion of plasmacytoid DCs (pDCs) among splenic DCs was (11.41 ± 3.82)% in the control group and (9.71 ± 3.22)% in the infection group at 2 weeks post-infection, and (4.26 ± 1.59)% and (4.85 ± 1.16)%, respectively, at 24 weeks post-infection, with no statistically significant differences (t = 0.760, -0.707; both P > 0.05). The proportion of conventional DCs (cDCs) among splenic DCs was (52.38 ± 5.13)% in the control group and (35.96 ± 4.31)% in the infection group at 2 weeks post-infection, with the infection group significantly lower than the control group (t = 5.479, P < 0.01); at 24 weeks post-infection, the proportions were (30.46 ± 5.44)% and (32.18 ± 3.22)%, respectively, with no statistically significant difference (t = -0.654, P > 0.05). Among splenic cDCs, the proportion of CD103⁺CD11b^- cells in the infection group was (14.24 ± 2.29)% at 2 weeks and (7.97 ± 1.75)% at 24 weeks post-infection, both significantly higher than those in the control group [(8.63 ± 0.60)% and (4.50 ± 1.28)%, respectively] (t = -5.294, -3.681; both P < 0.01). The proportion of CD103^-CD11b⁺ cells among splenic cDCs in the infection group was (45.74 ± 4.43)% at 2 weeks post-infection, significantly higher than that in the control group [(38.12 ± 4.58)%] (t = -2.672, P < 0.05), but was (50.42 ± 6.97)% at 24 weeks post-infection, significantly lower than that in the control group [(59.06 ± 4.51)%] (t = 2.378, P < 0.05). At 2 weeks post-infection, the proportions of CD40⁺ cells among splenic DCs, pDCs, and cDCs in the infection group were (13.89 ± 2.96)%, (2.33 ± 0.64)%, and (11.10 ± 1.93)%, respectively, all significantly lower than those in the control group [(23.98 ± 2.94)%, (3.75 ± 0.93)%, and (20.22 ± 2.84)%, respectively] (t = 5.412, 2.818, 5.943; P < 0.01, 0.05, 0.01). At 2 weeks post-infection, the proportions of CD80⁺ cells among splenic DCs and cDCs in the infection group were (27.78 ± 11.22)% and (19.36 ± 9.64)%, respectively, both significantly lower than those in the control group [(44.16 ± 8.11)% and (33.42 ± 7.61)%, respectively] (t = 2.645, 2.559; both P < 0.05). At 24 weeks post-infection, the proportions of CD80⁺ cells among splenic DCs and cDCs in the infection group were (47.22 ± 2.42)% and (31.00 ± 2.56)%, respectively, both significantly higher than those in the control group [(29.26 ± 7.88)% and (20.30 ± 7.45)%, respectively] (t = -5.338, -3.321; both P < 0.01). Conclusion In the early stage of E. multilocularis infection, the number of splenic DCs and cDCs decreased, and the expression of CD40 and CD80 was down-regulated, suggesting impaired DC maturation. In the late stage of infection, the number of DCs and cDCs increased, and CD80 expression was up-regulated, reflecting the evolution of the immune response during the late phase of infection.

Changes of splenic type 1 innate lymphoid cells subsets and their related factors expression in mice infected with Echinococcus multilocularis

XIAO Wenying, ABIDAN Ainiwaer, GE Conghui, TANG Na, SUN Sheng, WANG Mengying, GAO Yi, AYINAER Jiensi, HU Qiu, LI Jing, WANG Hui, ZHANG Chuanshan

2026, 44(2):  166-173.  doi:10.12140/j.issn.1000-7423.2026.02.003

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Objective To investigate the effects of Echinococcus multilocularis infection on the proportion of type 1 innate lymphoid cells (ILC1) and the expression levels of related factors in the spleen of mice. Methods C57BL/6J mice were randomly divided into the control group and the infection group. The infection group mice were injected with 3 000 E. multilocularis protoscoleces via the portal vein, while the control group mice were injected with the same volume of normal saline. After 24 weeks, spleen tissue sections were prepared from the infection group and the control group mice, and hematoxylin-eosin (HE) staining was used to observe the pathological changes in the spleen. Splenic lymphocytes were isolated from the mice, and flow cytometry was used to detect the proportion of ILC1 cell subsets, the expression of immune checkpoint molecules, and the levels of secreted cytokines in the spleen. Results HE staining showed that the boundary between the red pulp and the marginal zone of the white pulp was blurred in the spleen of mice infected with E. multilocularis, and hemosiderin deposition occurred in the white pulp area. Flow cytometry results indicated that the percentage of splenic natural killer (NK) cells among total splenic lymphocytes in the control group was (2.52 ± 0.44)%, which was higher than that in the infection group (1.25 ± 0.28)% (t = 6.42，P < 0.01). The percentage of splenic ILC1 among splenic NK cells in the control group was (20.07 ± 2.70)%, which was lower than that in the infection group (31.49 ± 3.28)% (t = 7.10, P < 0.01). The percentage of splenic conventional NK (cNK) cells among splenic NK cells in the infection group was (68.89 ± 3.28)%, which was lower than that in the control group (80.14 ± 3.49)% (t = 6.22, P < 0.01). The percentage of CD27^-CD11b⁺ cells among splenic ILC1 in the infection group was (21.37 ± 7.48)%, which was lower than that in the control group (43.49 ± 8.05)% (t = 5.33, P < 0.01); the percentages of CD27⁺CD11b^- and CD27⁺CD11b⁺ cells were (39.64 ± 8.80)% and (6.77 ± 1.51)%, respectively, which were higher than those in the control group (24.51 ± 8.35)% and (4.12 ± 1.71)% (t = 3.30, 3.06; both P < 0.01). The percentages of splenic ILC1 secreting programmed death receptor 1 (PD-1) and glucocorticoid-induced tumor necrosis factor receptor (GITR) in the infection group were (5.04 ± 1.54)% and (16.14 ± 4.53)%, respectively, which were higher than those in the control group (2.57 ± 1.52)% and (10.81 ± 4.03)% (t = 3.02, 2.33; both P < 0.05). The percentages of splenic ILC1 secreting granzyme B (Gr-B) and interferon-γ (IFN-γ) in the infection group were (6.16 ± 1.23)% and (16.13 ± 6.64)%, respectively, which were lower than those in the control group (9.42 ± 2.16)% and (30.68 ± 6.81)% (t = 2.94, 3.42; P < 0.05, 0.01). The percentage of splenic ILC1 secreting tumor necrosis factor-α (TNF-α) in the infection group was (22.26 ± 6.69)%, which was higher than that in the control group (10.80 ± 3.92)% (t = 3.31, P < 0.05). Conclusion In the late stage of E. multilocularis infection, the proportion of ILC1 cells and immature ILC1 cells were increased in mouse spleen. The co-inhibitory immune checkpoint molecule PD-1 was highly expressed, and the ability of these cells to secrete cytokines Gr-B and IFN-γ decreased. This suggested that ILC1 cell function suppressed might promote the chronic parasitism of E. multilocularis.

Dynamic changes of innate lymphoid cells in the liver of mice infected with Echinococcus multilocularis during the progression of hepatic fibrosis

CHEN Yuqing, SU Yaxin, WANG Ying, ZHOU Hao, CUI Lijun, JIANG Nan, ZHANG Jing, WANG Xiangqing, CAO Jianping, JIANG Bin, HUO Lele, SHEN Yujuan

2026, 44(2):  174-181.  doi:10.12140/j.issn.1000-7423.2026.02.004

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Objective To investigate the progression of hepatic fibrosis and the dynamic changes in the proportions of innate lymphoid cells (ILCs) and their subtypes in mice infected with Echinococcus multilocularis at various stages, and to examine the association between E. multilocularis-induced hepatic fibrosis and ILCs, so as to provide the theoretical basis for unraveling the immunoregulatory mechanisms underlying E. multilocularis-induced hepatic fibrosis. Methods BALB/c mice were randomly divided into an infection group and a control group (18 mice in each group). Each mouse in the infection group was injected with 1 500 E. multilocularis protoscoleces, and mice were euthanized 30, 90, and 180 days post-infection. Changes in the structure of liver tissues, inflammation and collagen deposition were observed using hematoxylin and eosin (HE) staining and Masson’s trichrome staining, and the expression of fibrosis-related proteins was quantified using immunofluorescence staining, including α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, Collagen Ⅰ, and Collagen Ⅲ. The proportion of ILCs in liver immune cells and the proportions of ILC subtypes (ILC1, ILC2, ILC3) in total ILCs were estimated using flow cytometry, and the relative IL-33 expression was quantified using quantitative Real-time PCR (qPCR) assay. The associations between ILC subtypes and fibrosis-related indicators were examined using Pearson correlation analysis. Results Vesicle-like lesions were seen in mouse livers 30 days post-infection with E. multilocularis, which gradually enlarged over time, and the lesions invaded surrounding liver tissues, leading to inflammation and even fibrotic lesions. HE staining showed aggravation of inflammation over time post-infection, and fiber spacing gradually formed 180 days post-infection. Masson’s trichrome staining showed higher proportions of collagen areas 90 days [(37.26 ± 7.83)% vs. (1.57 ± 1.17)%; t = 7.80, P < 0.01] and 180 days post-infection [(55.36 ± 8.21)% vs. (0.83 ± 0.73)%; t = 11.47, P < 0.01] than in controls. Immunofluorescence staining revealed higher ratios of the fluorescence intensity of α-SMA (1.08% vs. 0.51%; t = 4.62, P < 0.01), TGF-β1 (0.94% vs. 0.12%; t = 4.62, P < 0.01), Collagen Ⅰ (3.33% vs. 0.20%; t = 4.62, P < 0.01), and Collagen Ⅲ to 4', 6-diamidino-2-phenylindole (DAPI) (1.55% vs. 0.62%; t = 5.54, P < 0.01) in the infection group than in the control group, and flow cytometry showed a higher proportion of ILC in mouse liver leukocytes expressing CD45.2⁺ cells in the infection group than in the control group 30 days post-infection [(2.54 ± 0.43)% vs. (2.29 ± 0.31)%; t = 0.91, P > 0.05], a lower proportion of ILCs in the infection group than in the control group 90 days post-infection [(1.16 ± 0.51)% vs. (1.88 ± 0.31)%; t = 2.69, P < 0.05], and a higher proportion of ILCs in the infection group than in the control group 180 days post-infection [(0.56 ± 0.13)% vs. (0.31 ± 0.07)%; t = 3.02, P < 0.05]. The proportions of ILC1 in ILCs were lower in mice in the infection group than in the control group 90 [(61.22 ± 4.38)% vs. (75.96 ± 4.81)%; t = 5.067, P < 0.01] and 180 days post-infection [(11.11 ± 3.61)% vs. (47.50 ± 4.95)%; t = 10.29, P < 0.01], and negatively correlated with the ratio of collagen areas as revealed by Masson’s trichrome staining (r < 0, P < 0.05). The proportion of ILC2 in ILCs were higher in mice in the infection group than in the control group 180 days post-infection [(78.50 ± 4.10)% vs. (33.23 ± 4.57)%; t = 12.78, P < 0.01], and positively correlated with the ratio of collagen areas as revealed by Masson’s trichrome staining (r = 0.72, P < 0.05). There were no significant differences between the infection and control groups in terms of the proportion of ILC3 in ILCs 30 [(5.58 ± 2.37)% vs. (6.08 ± 0.83)%; t = 0.40, P > 0.05], 90 [(2.90 ± 0.84)% vs. (2.66 ± 0.68)%; t = 0.49, P > 0.05] and 180 days post-infection [(4.07 ± 2.94)% vs. (5.24 ± 3.74)%; t = 0.43, P > 0.05], and no significant associations were detected between the proportion of ILC3 in ILCs and fibrosis-related parameters. qPCR assay quantified no significant difference in the IL-33 mRNA expression in mouse liver tissues between the infection (1.37 ± 0.28) and control groups (1.03 ± 0.17) 30 days post-infection (t = 1.06, P > 0.05), higher IL-33 mRNA expression in the infection group (2.88 ± 0.49) than in the control group (0.74 ± 0.14) 90 days post-infection (t = 3.62, P < 0.05), and no significant difference in the IL-33 mRNA expression between the infection (1.55 ± 0.31) and control groups (1.06 ± 0.26) 30 days post-infection (t = 1.10, P > 0.05). Conclusion E. multilocularis infection may induce hepatic fibrosis in mice and alter the proportions of ILCs and their subtypes in hepatic immune cells. Notably, ILC2 expression is significantly upregulated at late stage of E. multilocularis infection, which may contribute to regulating hepatic stellate cell activation and promoting fibrosis.

Transcriptome analysis of splenic T follicular helper cells in mice infected with Schistosoma japonicum

LI Jiajia, WANG Xi, QIU Jiayin, ZHOU Xinjie, LV Chao, WANG Weisi, QIN Zhiqiang

2026, 44(2):  182-188.  doi:10.12140/j.issn.1000-7423.2026.02.005

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Objective To investigate the transcriptomics characteristics of splenic T follicular helper (Tfh) cells in mice infected with Schistosoma japonicum, and to screen and validate key differentially expressed genes (DEGs), so as to unravel the potential regulatory role of Tfh cells in the immune response to S. japonicum infection. Methods C57BL/6 mice were randomly divided into an infection group and a control group, of 8 mice each group. Mice in the infection group were infected with S. japonicum cercariae via skin contact, while animals in the control group received no treatment. Mouse spleens were sampled 6 weeks post-infection, and Tfh cells were isolated by gating based on the immune phenotype of positive cluster of differentiation 3, cluster of differentiation 4, CXC subfamily receptor 5, programmed death-1 (CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺) by means of negative selection with magnetic beads and flow cytometry. Total RNA was extracted from cell samples with an RNA extraction kit and purified. Bulk RNA-seq sequencing was performed on the Illumina platform to screen DEGs [|log₂FoldChange| > 1 and P < 0.05], and DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and screened DEGs were validated using reverse transcription quantitative Real-time PCR (RT-qPCR) assay. The expression of interleukin 5 (IL-5) and IL-13 proteins was measured using enzyme-linked immunosorbent assay (ELISA) in the serum of mice 2, 4, 6 and 8 weeks post-infection. Results Transcriptome sequencing identified a total of 1 379 DEGs, including 327 upregulated and 1 052 downregulated genes. GO and KEGG functional enrichment analyses revealed that these genes were significantly enriched in signal pathways of cytokine-cytokine receptor interaction, chemotaxis, and immune effector process regulation. RT-qPCR assay quantified higher relative il5 [(13.520 ± 1.540) vs. (1.060 ± 0.225); t = 10.52, P < 0.01] and il13 mRNA expression [(22.930 ± 0.720) vs. (1.137 ± 0.425); t = 28.41, P < 0.01] in the infection group than in the control group, and showed no significant difference between the infection and control groups in terms of relative neuromedin U receptor 1 (nmur1) [(11.210 ± 4.480) vs. (1.007 ± 0.084); t = 3.054, P > 0.05] or prostaglandin D2 receptor 2 (ptgdr2) mRNA expression [(4.405 ± 2.575) vs. (1.143 ± 0.443); t = 1.629, P > 0.05]. ELISA measured extremely low IL-5 (median, 2.00 pg/mL) and IL-13 concentrations (median, 4.00 pg/mL) in both uninfected mice and at early stages of infections (2 weeks post-infection). The median IL-13 concentration increased to 68.20 pg/mL at 4 weeks post-infection (P < 0.05), and the median IL-5 concentration rose to 77.06 pg/mL at 6 weeks post-infection (P < 0.05), which was maintained a high level in subsequent infection phases. Conclusion S. japonicum infection remarkably reshapes the transcriptome profile of murine splenic Tfh cells, leading to a highly activation state. Tfh cells may participate in maintaining the host Th2-type immune microenvironment and regulating immunopathology through significantly upregulating il5 and il13 expression.

Transcriptome analysis of liver tissues from Schistosoma japonicum-infected mice with tigit knockout natural killer cells

PENG Hui, CUI Lijun, ZHANG Xiaocheng, ZHANG Jing, YIN Jianhai, LU Shaohong, CAO Jianping

2026, 44(2):  189-195.  doi:10.12140/j.issn.1000-7423.2026.02.006

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Objective To investigate the effect of conditional knockout of inhibitory receptor T-cell immunoreceptor immunoglobulin and tyrosine-based inhibitory motif domain (tigit) in natural killer (NK) cells on disease progression in a model of schistosomiasis liver fibrosis and unravel the underlying mechanisms. Methods Four mice with tigit conditional knockout NK cells were used as the experimental group, and four wild-type mice in the same litter served as controls. All mice were percutaneously infected with Schistosoma japonicum cercariae (20 ± 2 per mouse), and mouse liver tissues were sampled 6 weeks post infection. The morphological structure of egg granulomas, degree of inflammatory cell infiltration, and collagen fiber deposition were observed using hematoxylin and eosin (HE) staining and Masson’s trichrome staining, and the degree of liver fibrosis was assessed using immunohistochemical staining of α smooth muscle actin (α-SMA). The percentage of positively stained area was calculated using the software ImageJ. The relative mRNA expression of alpha smooth actin (acta2), Ⅰ-collagen (I-c) and tigit was quantified with a reverse transcription quantitative Real-time PCR (RT-qPCR) assay. Total RNA extracted from mouse liver tissues was subjected to transcriptome sequencing to identify differentially expressed genes, followed by Gene Ontology (GO) functional enrichment analysis. All statistical analyses were performed using the software GraphPad Prism 9, and differences of means were tested for statistical significance with independent samples t test. Results HE staining revealed smaller areas of egg granulomas and milder inflammatory cell infiltration in the experimental group relative to the control group. Masson’s trichrome staining showed reduced collagen deposition that was confined to localized areas and presented as slender fibrous strands in mouse liver tissues in the experimental group, and deposition of extensive blue collagen fibers in mouse liver tissues in the control group, producing obvious fibrous encapsulation around egg granulomas. The score for collagen deposition was significantly lower in the experimental group (8.07 ± 0.52 points) than in the control group (26.07 ± 1.54 points) (t = 19.22, P < 0.01). Immunohistochemical staining for α-SMA revealed a marked decrease in positive α-SMA expression and staining intensity in mouse liver tissues in the experimental group, whereas widespread positive α-SMA expression was detected in mouse liver tissues in the control group, which was mainly found around activated hepatic stellate cells around granulomas and in the perisinusoidal spaces. Semi-quantitative analysis revealed a lower percentage of α-SMA positive areas in the experimental group (3.39 ± 1.25) than in the control group (8.60 ± 2.47) (t = 5.92, P < 0.01). RT-qPCR assay quantified lower relative mRNA expression of the fibrosis-related genes acta2 [(0.87 ± 0.06) vs. (1.40 ± 0.03); t = 14.93, P < 0.01], I-c [(0.88 ± 0.03) vs. (1.2 ± 0.02); t = 14.99, P < 0.01], and tigit [(1.67 ± 0.15) vs. (1.03 ± 0.11); t = 6.216, P < 0.01] in the experimental group than in the control group. Transcriptome sequencing identified 431 differentially expressed genes (365 up-regulated and 66 down-regulated genes), and GO enrichment analysis indicated that the up-regulated genes were significantly enriched in immune response-related pathways, including pathways of NK cell degranulation, negative regulation of hepatic stellate cell activation, and neutrophil chemotaxis and migration. Conclusion Conditional knockout of tigit in NK cells may alleviate schistosomiasis liver fibrosis in mice by restoring NK cell functions and inhibiting hepatic stellate cell activation, and affect the neutrophil pathway, suggesting that TIGIT has a complex regulatory role in the liver immune microenvironment.

Transcriptome analysis of splenic B cells in mice infected with Schistosoma japonicum

QIU Jiayin, LI Jiajia, ZHOU Xinjie, LI Yinlong, FENG Ting, ZHENG Mao, LV Chao, QIN Zhiqiang

2026, 44(2):  196-202.  doi:10.12140/j.issn.1000-7423.2026.02.007

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Objective To analyze the transcriptomic characteristics of splenic B cells in mice infected with Schistosoma japonicum, screen and validate differentially expressed genes (DEGs), and provide a basis for elucidating the regulatory mechanisms of B cells during S. japonicum infection. Methods Balb/C mice were randomly divided into the control group (NC group), the 4-week post-infection group (Sj_4W group), and the 6-week post-infection group (Sj_6W group). Mice in the Sj_4W and Sj_6W groups were infected percutaneously using the patch method with (25 ± 1) S. japonicum cercariae. Mice were sacrificed at week 4 and 6 post-infection and spleen tissues were collected, respectively. Splenic B cells were isolated and enriched by magnetic bead sorting. Total RNA was extracted from splenic B cells, and transcriptome libraries were constructed. RNA sequencing was performed on the Illumina platform. Differential expression analysis was conducted using the limma and edgeR packages to screen for DEGs. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and gene ontology (GO) functional enrichment classification were performed on the DEGs. Three highly expressed genes were selected for validation by reverse transcription quantitative Real-time PCR (RT-qPCR). Results Cluster analysis revealed that a total of 6 443 DEGs were identified between the Sj_6W and NC groups, including 4 421 up-regulated genes and 2 022 down-regulated genes. A total of 6 757 DEGs were identified between the Sj_6W and Sj_4W groups, including 4 754 up-regulated genes and 2 003 down-regulated genes. GO functional enrichment analysis showed that downregulated genes were primarily involved in lymphocyte differentiation, immune response regulatory signaling pathways, and lymphocyte proliferation, whereas upregulated genes were mainly enriched in lymphocyte-mediated immunity, production of immune response molecular mediators, B cell-mediated immunity, and humoral immunity. KEGG pathway enrichment analysis revealed that downregulated genes were mainly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, Th1 and Th2 cell differentiation, and Th17 cell differentiation, whereas upregulated genes were mainly enriched in cytokine-cytokine receptor interaction, extracellular matrix-receptor interaction, and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. RT-qPCR validation confirmed that the relative mRNA expression levels of interleukin-27 (IL-27), matrix metalloproteinase-8 (MMP-8), and eosinophil protein X (EPX) in splenic B cells of the Sj_6W group were 2.14 ± 1.18, 21.86 ± 14.06, and 324.40 ± 252.40, respectively, which were significantly higher than those in the Sj_4W group (0.31 ± 0.18, 0.03 ± 0.02, and 0.44 ± 0.72; Mann-Whitney U test, P < 0.01) and also higher than those in the NC group (1.04 ± 0.29, 1.20 ± 0.72, and 1.17 ± 0.69; Mann-Whitney U test, P < 0.05). Conclusion Following S. japonicum infection, splenic B cells in mice underwent transcriptional reprogramming, with upregulated expression of IL-27, EPX, and MMP-8, accompanied by alterations in pathways related to lymphocyte differentiation and proliferation, Th1/Th2 and Th17 cell differentiation, production of immune response molecular mediators, B cell-mediated immunity and humoral immune regulation, cytokine-cytokine receptor interaction, and extracellular matrix-receptor interaction.

Preparation and application of polyclonal antibodies against endoplasmic reticulum stress-related proteins TgBip and TgeIF2α of Toxoplasma gondii

TIAN Siyu, MOU Yani, TAN Feng

2026, 44(2):  203-208.  doi:10.12140/j.issn.1000-7423.2026.02.008

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Objective To prepare polyclonal antibodies against Toxoplasma gondii immunoglobulin heavy chain-binding protein (TgBip) and eukaryotic translation initiation factor 2α (TgeIF2α), and to evaluate their specificity. Methods Specific primers for amplification of TgBip and TgeIF2α were designed using bioinformatics methods. Target genes were amplified using PCR assay with cDNA from tachyzoites of T. gondii RH strain as a template. The recombinant plasmids pColdⅢ-His-TgBip and pColdⅢ-His-TgeIF2α were constructed using in-fusion cloning, transformed into Escherichia coli BL21 competent cells. Following induction of protein expression with isopropyl-β-D-thiogalactoside (IPTG), the expression of recombinant proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting assay, and proteins were purified using Ni-NTA affinity chromatography. New Zealand white rabbits were immunized with purified TgBip and TgeIF2α proteins as antigens to prepare specific anti-TgBip and anti-TgeIF2α polyclonal antibodies. The recognition of endogenous TgBip and TgeIF2α proteins by polyclonal antibodies was analyzed using enhanced chemiluminescence assay. Changes in TgBip protein expression were examined using indirect immunofluorescence assay (IFA) during the process of endoplasmic reticulum stress, and changes in TgeIF2α phosphorylation levels were detected using Western blotting assay during the process of endoplasmic reticulum stress. Results PCR assay showed that the specific amplification fragments of TgBip and TgeIF2α were 1 920 and 1 044 bp in size, respectively, which were consistent with expected fragment sizes. The recombinant plasmids pColdⅢ-His-TgBip and pColdⅢ-His-TgeIF2α were successfully constructed. SDS-PAGE and Western blotting assay showed high expression of TgBip and TgeIF2α proteins in BL21 competent cells, with relative molecular masses (M_r) of approximately 71 000 and 40 000, which approached to the theoretical values. Western blotting assay showed that the prepared antibodies would specifically recognize endogenous T. gondii TgBip and TgeIF2α proteins, with no cross-reactions to host cells. IFA showed that TgBip was distributed in T. gondii endoplasmic reticulum, and TgBip was distributed around the cell nucleus prior to induction of endoplasmic reticulum stress and dispersed following induction of endoplasmic reticulum stress. Western blotting assay determined higher relative expression of phosphorylated TgeIF2α following induction of endoplasmic reticulum stress than prior to induction of endoplasmic reticulum stress [(2.199 ± 0.376) vs. (1.217 ± 0.099); t = 4.379, P < 0.05]. Conclusion The prepared specific polyclonal antibodies against TgBip and TgeIF2α may be used to detect the endoplasmic reticulum structure and stress level of T. gondii.

Impact of Toxoplasma gondii infection on mucosal immunopathology in mouse small intestines

ZHANG Ling, LIN Xincheng, LV Fangli

2026, 44(2):  209-213.  doi:10.12140/j.issn.1000-7423.2026.02.009

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Objective To examine the effect of acute Toxoplasma gondii infection on mucosal immune responses and immunopathology in mouse small intestines and unravel the mechanism. Methods Fifteen Kunming mice were randomly divided into two groups, including the T. gondii infection group (10 mice) and the control group (5 mice). Mice in the infection group were intraperitoneally injected with tachyzoites of the T. gondii RH strain (1 × 10³ tachyzoites per mouse), while animals in the control group were injected with an equal volume of PBS. Mice were sacrificed at 7 days post-infection, and the posterior section of small intestine (2 cm in length) and mesenteric lymph nodes were taken for paraffin embedding, preparing slices, and hematoxylin and eosin staining to observe the pathological changes of mice. The intestinal villi length and the crypt depth were measured, and the numbers of goblet cells and Paneth cells were counted. In addition, immunohistochemical staining was performed to count the number of CD68⁺ cells in small intestines. Differences of means were tested for statistical significance using independent sample t test with the software GraphPad Prism 10.1.2. Results The structures of mouse small intestinal villi and mesenteric lymph nodes were intact with a normal morphology in the control group, while mice in the infection group exhibited severe pathological damage in the small intestine, characterized by necrosis of villous epithelial cells, shedding or shortening of intestinal villi, deepening of intestinal crypts, and extensive infiltration of inflammatory cells in small intestinal mucosal tissues. Additionally, the histological structures of the mesenteric lymph nodes of mice in the infection group were disrupted, with diffuse necrotic lesions observed. T. gondii tachyzoites and pseudocysts were observed in mouse small intestines and mesenteric lymph nodes in the infection group. The mean length of mouse small intestinal villi was shorter in the infection group than that in the control group [(93.07 ± 2.158) μm vs. (125.0 ± 19.36) μm; t = 2.756, P < 0.05], and the mean depth of mouse small intestinal crypts was greater in the infection group than that in the control group [(49.04 ± 8.809) μm vs. (39.9 ± 4.174) μm; t = 2.382, P < 0.05]. The number of goblet cells per 10 mouse small intestinal villi was lower in the control group than that in the infection group [(78.70 ± 14.01) cells vs. (96.71 ± 13.89) cells; t = 2.618, P < 0.05], and the number of goblet cells per 10 mouse small intestinal crypts was fewer in the infection group than that in the control group [(31.60 ± 8.60) cells vs. (53.17 ± 13.03) cells; t = 4.014, P < 0.05]. The number of Paneth cells per 20 mouse small intestinal crypts was fewer in the infection group than that in the control group [(69.80 ± 8.90) cells vs. (103.0 ± 4.86) cells; t = 8.342, P < 0.01]. In addition, the number of CD68⁺ macrophages per high-power field in mouse small intestine tissue was larger in the infection group than that in the control group [(42.33 ± 11.41) cells vs. (15.75 ± 2.754) cells; t = 4.496, P < 0.01], and the number of CD68⁺ macrophages in mouse Peyer’s patches per high-power field was higher in the infection group than that in the control group [(185.0 ± 23.80) cells vs. (15.75 ± 2.754) cells; t = 9.286, P < 0.01]. Conclusion Acute T. gondii infection may induce small intestinal damage in mice, with a decrease in goblet cell and Paneth cell counts and an increase in CD68⁺ macrophage number in small intestinal tissues, suggesting that goblet cells, Paneth cells, and macrophages may contribute to mucosal immune responses and immunopathology of small intestines.

Effect of Toxoplasma gondii infection in the first trimester on Tim-3 expression in decidual immune cells and differential analysis

ZHANG Han, YANG Ruohan, CHEN Zihuan, ZHANG Haixia, FENG Xiaoyu, WEI Dianfang, WANG Wenxiao, LIU Xianbing, HU Xuemei

2026, 44(2):  214-221.  doi:10.12140/j.issn.1000-7423.2026.02.010

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Objective To investigate the effect of Toxoplasma gondii infection during pregnancy on the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in decidual immune cells and differential relationships. Methods Pregnant mice were assigned into the wild-type (WT) pregnant control group, the WT pregnant infection group, and the Tim-3^-/- pregnant infection group, of 20 mice each group. Mice in two infection groups were intraperitoneally inoculated with 250 tachyzoites of the T. gondii RH strain suspended in 250 μl of diluents at gestational ages of 8 days, while mice in the control group received an equal volume of physiological saline. The mental state of pregnant mice was observed in each group at gestational ages of 14 days, and all pregnant mice were sacrificed with euthanasia, dissected, recorded for pregnancy outcomes and calculate for the proportion of abnormal fetuses. Placentas and fetuses were harvested from pregnant mice and weighed, and the differences in adverse pregnancy outcomes were compared among three groups. Decidual tissues were collected from healthy pregnant women undergoing voluntary termination of pregnancy in the first trimester, and decidual mononuclear cells were isolated using density gradient centrifugation and divided into a control group and an infection group, with 1 × 10⁷ cells per group. Cells in the infection group were challenged with T. gondii at a cell-to-parasite ratio of 3 ∶ 1, whereas cells in the control group were treated with an equal volume of T. gondii-free culture media. Cells in two groups were harvested 20 hours post-incubation, and the expression of Tim-3 was detected on decidual natural killer (dNK) cells, decidual macrophages (dMϕ), decidual myeloid-derived suppressor cells (dMDSCs), and decidual dendritic cells (dDCs) using flow cytometry. dNK cells, dMϕ, dMDSCs and dDCs were further sorted from decidual mononuclear cells with immunomagnetic beads, and each cell type was subdivided into an infection subgroup and a control subgroup. Cells in the infected subgroups were infected with T. gondii at a cell-to-parasite ratio of 3∶1, and those in control subgroups were incubated with an equal volume of parasite-free culture media. Cells were harvested 20 hours post-incubation for protein purification, and Tim-3 expression in each type of decidual immune cells was determined using Western blotting assay. Between-group differences were tested for statistical significance with independent-sample t-test for in vivo experiments or paired-sample t-test for in vitro experiments. Results The placental [(83.13 ± 9.13) mg vs. (105.18 ± 9.64) mg; t = 4.394, P < 0.01] and fetal weights [(96.81 ± 11.63) mg vs. (122.86 ± 3.67) mg; t = 5.655, P < 0.01] were significantly lower in the WT infection group than in the WT control group, and the placental [(66.31 ± 9.00) mg vs. (83.13 ± 9.13) mg; t = 3.471, P < 0.01] and fetal weights [(74.32 ± 6.33) mg vs. (96.81 ± 11.63) mg; t = 4.495, P < 0.05] were significantly lower in the Tim-3^-/- infection group than in the WT infection group. No aborted fetuses were observed in the WT control group, and the abortion rate was significantly higher in the WT infection group [(30.14 ± 9.87)%] than that in the WT control group (t = 8.076, P < 0.01), while the abortion rate was higher in the Tim-3^-/- infection group [(66.85 ± 26.17)%] than in the WT infection group (t = 3.473, P < 0.01). Adverse pregnancy outcomes were significantly aggravated in pregnant mice in the Tim-3^-/- infection group than in the WT infection group. Flow cytometry measured lower percentages of Tim-3 positive cells in human dNK cells [(12.22 ± 4.61)% vs. (24.50 ± 7.10)%; t = 7.717, P < 0.05], dMϕ [(23.87 ± 5.26)% vs. (41.23 ± 3.84)%; t = 5.865, P < 0.05], dMDSCs [(10.07 ± 1.02)% vs. (17.53 ± 0.65)%; t = 7.798, P < 0.05] and dDCs [(11.01 ± 4.10)% vs. (18.53 ± 4.93)%; t = 9.592, P < 0.05] in the infection group than in the control group, and Western blotting assay determined lower relative Tim-3 expression in human dNK cells [(0.69 ± 0.09) vs. (1.03 ± 0.06); t = 7.562, P < 0.01], dMϕ [(0.74 ± 0.09) vs. (0.98 ± 0.06); t = 4.099, P < 0.05], dMDSCs [(0.79 ± 0.08) vs. (0.95 ± 0.02); t = 4.398, P < 0.05], and dDCs [(0.84 ± 0.04) vs. (0.97 ± 0.06); t = 5.455, P < 0.05] in the infection group than in the control group, and flow cytometry measured the largest reduction [(50.82 ± 5.92)%] in Tim-3 expression in dNK cells following T. gondii infections, with (42.37 ± 7.96)% and (42.08 ± 12.31)% reductions in dMDSCs and dMϕ, and the smallest decrease in dDC cells [(41.45 ± 7.64)%]. Like flow cytometric analysis, Western blotting assay determined a reduction in Tim-3 expression in all types of decidual immune cells following T. gondii infections, with the largest reduction in dNK cells [(33.06 ± 8.52)%], followed by in dMϕ [(22.84 ± 10.64)%], and mild reductions in dMDSCs [(16.25 ± 7.44)%] and dDCs [(13.6 ± 4.42)%]. Conclusion T. gondii infections in the first trimester significantly downregulates Tim-3 expression in major decidual immune cell subsets at the maternal-fetal interface, with the most pronounced effect on dNK cells, followed by dMϕ, dMDSCs, and dDCs. Downregulation of Tim-3 expression in various types of decidual immune cells following T. gondii infection may play an important role in the development of adverse pregnancy outcomes.

Biological functions of enolase 1 in modulation of Toxoplasma gondii tachyzoite behaviors and tachyzoite-bradyzoite transition

ZHU Di, WU Weiling, KONG Dehao, ZHOU Zhihao, PENG Hongjuan

2026, 44(2):  222-228.  doi:10.12140/j.issn.1000-7423.2026.02.011

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Objective To investigate the specific biological functions of Toxoplasma gondii bradyzoite-specific enolase 1 (ENO1) in the tachyzoite-bradyzoite transition, and to analyze the role and molecular mechanism of the ENO family in the regulatory network of T. gondii stage transition. Methods The eno1-deleted strain ME49-Δeno1 and two complementary strains ME49-Δeno1-Comp-eno1 and ME49-Δeno1-Comp-eno2 were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing technology. The growth, proliferation, and invasion of four T. gondii strains (ME49-Δku80, ME49-Δeno1, ME49-Δeno1-Comp-eno1, and ME49-Δeno1-Comp-eno2) were evaluated following infections in human foreskin fibroblast cells with the plaque assay, proliferation assay, and invasion assay, respectively. The relative transcriptional levels of tachyzoite-specific surface antigen 1 (sag1) and bradyzoite-specific antigen 1 (bag1) genes were quantified in ME49-Δku80 and ME49-Δeno1 strains using quantitative Real-time PCR (qPCR) assay under in vitro alkaline induction to evaluate the effect of eno1 deletion on the ability of T. gondii tachyzoites transition to bradyzoites, and the synthesis of nascent peptide chains was detected in ME49-Δku80 and ME49-Δeno1 strains using puromycin labeling and Western blotting assay to examine the effect of eno1 deletion on the synthesis of T. gondii proteins. Results The eno1-deleted strain ME49-Δeno1 and its two complementary strains ME49-Δeno1-Comp-eno1 and ME49-Δeno1-Comp-eno2 were successfully generated. Plaque, proliferation, and invasion assays showed that the areas of plaques were (16 538 ± 14 310), (4 376 ± 5 355), (17 377 ± 14 333), and (8 710 ± 8 207) μm², and the average numbers of parasites per parasitophorous vacuole were (5.836 ± 0.382), (4.792 ± 0.150), (5.165 ± 1.243), and (3.872 ± 0.301) in ME49-Δku80, ME49-Δeno1, ME49-Δeno1-Comp-eno1, and ME49-Δeno1-Comp-eno2 strains, while the invasion efficiencies of ME49-Δku80, ME49-Δeno1, ME49-Δeno1-Comp-eno1, and ME49-Δeno1-Comp-eno2 strains were (57.802 ± 6.932)%, (19.679 ± 4.508)%, (63.833 ± 1.198)%, and (35.901 ± 8.098)%, respectively, indicating that deletion of eno1 inhibited the growth (F = 38.290, P < 0.01), intracellular proliferation (F = 4.467, P < 0.05), and invasion ability (F = 36.650, P < 0.01) of T. gondii. Complementation of eno1 fully restored these abilities, and there were no significant differences between ME49-Δeno1-Comp-eno1 and ME49-Δku80 in terms of the plaque areas (t = 0.459, P > 0.05), average number of T. gondii tachyzoites per parasitophorous vacuole (t = 0.895, P > 0.05), or invasion efficiency (t = 1.485, P > 0.05). Nevertheless, complementation of eno2 partially restored the growth, proliferation and invasion abilities of T. gondii. The area of plaques generated by the ME49-Δeno1-Comp-eno2 strain was larger than that by the ME49-Δeno1 strain (t = 5.263, P < 0.05), but smaller than that by the ME49-Δku80 strain (t = 4.905, P < 0.01), and the average number of tachyzoites per parasitophorous vacuole was lower in the ME49-Δeno1-Comp-eno2 strain than in the ME49-Δku80 strain (t = 6.998, P < 0.05), while the invasion efficiency of the ME49-Δeno1-Comp-eno2 strain was higher than that of the ME49-Δeno1 strain (t = 3.032, P < 0.05) but lower than that of the ME49-Δku80 strain (t = 3.559, P < 0.05). Following alkaline induction for 48 h, there was no significant difference in the relative transcriptional level of sag1 gene between the ME49-Δeno1 strain (1.067 ± 0.115) and the ME49-Δku80 strain (1.002 ± 0.154) (t = 0.584, P > 0.05), and the relative transcriptional level of bag1 gene was lower in the ME49-Δeno1 strain (13.172 ± 1.679) than in the ME49-Δku80 strain (20.338 ± 1.344) (t = 5.770, P < 0.01). Following alkaline induction for 96 h, the relative transcriptional level of sag1 gene was higher in the ME49-Δeno1 strain (0.957 ± 0.132) than in the ME49-Δku80 strain (0.727 ± 0.053) (t = 2.803, P < 0.05), and the relative transcriptional level of bag1 gene was lower in the ME49-Δeno1 strain (162.248 ± 23.377) than in the ME49-Δku80 strain (231.413 ± 12.910) (t = 4.486, P < 0.05). Western blotting assay detected no significant difference in the level of nascent peptide synthesis between the ME49-Δku80 and ME49-Δeno1 strain, indicating no significant effect of eno1 deletion on the synthesis ability of T. gondii proteins. Conclusion Bradyzoite-specific enolase eno1 promotes the growth of T. gondii tachyzoites and positively regulates the transition from tachyzoites to bradyzoites. It is a key regulator but not compulsory in the T. gondii tachyzoite-bradyzoite transition. In addition, ENO1 cannot be functionally replaced by ENO2 in the proliferation of T. gondii tachyzoites.

Effect of Toxoplasma gondii infection during the first trimester on expression of the immune inhibitory molecule LAG-3 on decidual T cell surface

YANG Ruohan, ZHANG Han, REN Liqin, LV Linyao, WEI Dianfang, FENG Xiaoyu, WANG Wenxiao, LIU Xianbing, HU Xuemei

2026, 44(2):  229-236.  doi:10.12140/j.issn.1000-7423.2026.02.012

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Objective To investigate the effect of Toxoplasma gondii infection on the expression of lymphocyte-activation gene 3 (LAG-3) and related functional molecules on the surface of decidual T cells at the maternal-fetal interface, and to examine its potential association with adverse pregnancy outcomes. Methods Mononuclear cells were isolated from healthy individuals’ decidual tissues in the first trimester and divided into three groups, including the control group, T. gondii infection group, and infection + LAG-3 inhibitorr (10 μmol/L Relatlimab) group, of 1.0 × 10⁷ cells each group. Cells in the control group were given no treatments, while cells in the infection and infection + LAG-3 inhibitor groups were infected with T. gondii tachyzoites at a tachyzoite-to-cell ratio of 1 ∶ 3 and co-cultured in vitro for 19 h prior to cell harvesting. The expression of LAG-3 and intracellular functional molecules interferon gamma (IFN-γ) and granzyme B was detected on the surface of decidual CD4⁺ T (dCD4⁺ T) and CD8⁺ T (dCD8⁺ T) cells using flow cytometry. Human dCD4⁺ and dCD8⁺ T cells were isolated from aforementioned mononuclear cells with CD4/CD8 sorting magnetic beads and purified, and assigned into the control and T. gondii infection groups to quantify LAG-3, IFN-γ and granzyme B expression in presence of T. gondii infection alone. Human dCD4⁺ and dCD8⁺ T cells were divided into the infection and infection + LAG-3 inhibitor groups to examine the effect of T. gondii infection on IFN-γ and granzyme B expression in decidual T cells following blockade of LAG-3. Cells in the control group were given no treatments, while cells in the infection and infection + LAG-3 inhibitor groups (1.0 × 10⁷ cells each group) were infected with T. gondii tachyzoites at a tachyzoite-to-cell ratio of 1 ∶ 3 and co-cultured in vitro for 19 h prior to cell harvesting. Total protein was extracted from cells, and the expression of LAG-3, IFN-γ, and granzyme B proteins was determined in dCD4⁺ and dCD8⁺ T cells using Western blotting assays. All statistical analyses were performed using the software GraphPad Prism 10.3.0. Differences of means between groups were tested for statistical significance with independent-sample Student’s t-test. Results Flow cytometry detected lower proportions of LAG-3-positive human dCD4⁺ [(17.30 ± 1.50)% vs. (27.13 ± 1.45)%; t = 19.750, P < 0.01] and dCD8⁺ T cells [(22.20 ± 2.98)% vs. (35.50 ± 3.60)%; t = 6.389, P < 0.05] in the infection group than those in the control group, higher proportions of IFN-γ-positive human dCD4⁺ T [(7.15 ± 6.90)% vs. (5.46 ± 0.47)%; t = 6.230, P < 0.05] and dCD8⁺ T cells [(3.94 ± 0.27)% vs. (3.02 ± 0.48)%; t = 7.101, P < 0.05] in the infection group than in the control group, higher proportions of IFN-γ-positive human dCD4⁺ T [(8.51 ± 0.36)% vs. (7.15 ± 6.90)%; t = 13.350, P < 0.01] and dCD8⁺ T cells [(5.83 ± 1.01)% vs. (3.94 ± 0.27)%; t = 4.73, P < 0.05] in the infection + LAG-3 inhibitor group than in the infection group. Similarly, higher percentages of granzyme B-positive human dCD4⁺ [(78.30 ± 1.01)% vs. (72.30 ± 1.5)%; t = 8.260, P < 0.05] and dCD8⁺ T cells [(49.00 ± 6.44)% vs. (39.12 ± 10.25)%; t = 5.164, P < 0.05] were detected in the infection group than in the control group, and higher proportions of granzyme B-positive human dCD4⁺ (87.00% ± 0.65%) and dCD8⁺ T cells (57.30% ± 3.93%) were seen in the infection + LAG-3 inhibitor group than in the infection group (t = 18.710, P < 0.01; t = 5.636, P < 0.05). Western blotting assay determined lower relative LAG-3 protein expression in human primary dCD4⁺ [(0.89 ± 0.06) vs. (1.09 ± 0.04); t = 10.790, P < 0.01] and dCD8⁺ T cells [(0.78 ± 0.06) vs. (0.99 ± 0.10); t = 4.503, P < 0.05] in the infection group than in the control group, and higher relative IFN-γ and granzyme B protein expression in dCD4⁺ [IFN-γ: (0.85 ± 0.05) vs. (0.64 ± 0.04), t = 16.77, P < 0.01; granzyme B: (0.75 ± 0.05) vs. (0.61 ± 0.09), t = 5.141, P < 0.05] and dCD8⁺ T cells [IFN-γ: (0.94 ± 0.08) vs. (0.66 ± 0.07), t = 6.193, P < 0.05; granzyme B: (0.80 ± 0.08) vs. (0.44 ± 0.09), t = 4.446, P < 0.05] in the infection group than in the control group. Similarly, higher IFN-γ and granzyme B protein expression was determined in dCD4⁺ [IFN-γ: (0.97 ± 0.06) vs. (0.77 ± 0.08), t = 13.940, P < 0.01; granzyme B: (0.96 ± 0.04) vs. (0.70 ± 0.08), t = 10.250, P < 0.01] and dCD8⁺ T cells [IFN-γ: (1.15 ± 0.11) vs. (0.86 ± 0.07), t = 5.346, P < 0.05; granzyme B: (1.03 ± 0.30) vs. (0.78 ± 0.14), t = 10.520, P < 0.01] in the infection + LAG-3 inhibitor group than in the infection group. Conclusion T. gondii infection in the first trimester may down-regulate the expression of LAG-3 on the surface of dCD4⁺ T and dCD8⁺ T cells, thereby up-regulating the expression of functional molecules IFN-γ and granzyme B, which may be an important mechanism responsible for adverse pregnancy outcomes.

Establishment and application of high-throughput screening platform for anti-Babesia compounds

ZHANG Yuting, BAI Yanan, YIN Hong, GUAN Guiquan, MA Yonghua, WANG Jinming

2026, 44(2):  237-243.  doi:10.12140/j.issn.1000-7423.2026.02.013

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Objective To establish a high-throughput screening system for anti-Babesia compounds to identify highly active candidate compounds, and to evaluate the in vivo efficacy of these compounds against Babesia infections in animal models. Methods Specific primers were designed targeting B. duncani histone-like transcription factor gene (histf; GenBank accession number: XM_067945812). A B. duncani culture with an initial parasitemia of 21.48% was subjected to serial dilutions to prepare 7 samples with different parasitemia levels (2.148 × 10^-1 to 1.375 × 10^-5). Genomic DNA was extracted from each sample, and the histf gene was amplified by quantitative Real-time PCR (qPCR) assay to construct a standard curve correlating parasite burden with cycle threshold (Ct) values. B. duncani-infected erythrocytes were seeded onto 96-well plates (5.5 × 10⁴ cells/well), followed by addition of 1 212 compounds from the Anti-Infection Compound Library at a final concentration of 1 × 10^-5 mol/L to evaluate the anti-parasitic activity. The 20 most active compounds were serially diluted (1 × 10^-7 to 3 × 10^-5 mol/L) and incubated for 48 h and 72 h, respectively. Genomic DNA was then extracted from parasites in each group for amplification of the histf gene using qPCR assay to yield Ct values, and the inhibitory rates and in vitro half-maximal inhibitory concentrations (IC₅₀) were calculated. HEK-293T cells were seeded onto 96-well plates at 8 000 cells per well. The 20 most active compounds were serially diluted into concentrations of 1 × 10^-6, 3 × 10^-6, 1 × 10^-5, 3 × 10^-5, 1 × 10^-4, and 3 × 10^-4 mol/L and added to respective wells, and a vehicle control group (without compound treatment) was also assigned. Cell viability was assessed using the CCK-8 assay by measuring the absorbance at 450 nm (A₄₅₀ value), and the half-maximal cytotoxic concentration (CC₅₀) were calculated. Twenty golden hamsters and 12 BALB/c mice were intraperitoneally inoculated with B. duncani-infected erythrocytes (1 × 10² cells/animal) and B. microti-infected erythrocytes (1 × 10⁴ cells/animal), and then randomly assigned to control and treatment groups. Animals in treatment groups were intraperitoneally injected with dacinostat at a dose of 10 mg/kg 2 days post-infection for 15 consecutive days, while animals in controls groups were given the same volume of vehicles. Giemsa-stained blood smears were prepared daily, and erythrocyte numbers were counted and parasitemia was determined to evaluate in vivo efficacy. All statistical analyses were performed using the software GraphPad Prism 10.1.2, and differences of means between groups were tested for statistical significance with unpaired t test. Results qPCR amplification showed normal amplification curves using histf as the target gene, and the melting curve exhibited a single peak, indicating a stable qPCR system and highly specific amplification products. Ct values showed a good linear correlation with parasitemia, with a standard curve slope of ‒3.734, a coefficient of determination (R²) of 0.993, and a regression equation of y = ‒3.734x + 32.272. Standard curve analysis showed that Ct values presented a tendency towards a rise with a reduction in parasitemia. Screening of 1 212 compounds from the compound library showed inhibitory rates of ‒47.1% to 98.1% against B. duncani, including 31 compounds with inhibitory rates of > 80%, and 20 compounds showed in vitro inhibitory activity against B. duncani, with IC₅₀ values ranging from 3.3 × 10^-8 to 1.325 × 10^-5 mol/L. Dacinostat showed the strongest inhibitory activity, with an IC₅₀ of 3.3 × 10^-8 mol/L. The cytotoxicity of each compound against HEK-293T cells increased over time, and six compounds, including dacinostat, camostat mesylate and fenchol, exhibited both high anti-parasitic activity and low cytotoxicity. In golden hamster models of B. duncani infections, dacinostat delayed parasite detection and significantly reduced parasitemia relative to vehicles 12 days post-infection [(1.75 ± 0.94)% vs. (17.24 ± 3.15)%; t = 4.798, P < 0.05]. Two animals survived at the end of the experiment, while all animals died in the control group. In BALB/c mouse models of B. microti infections, dacinostat also significantly reduced parasitemia relative to vehicles 15 days post-infection [(0.06 ± 0.02)% vs. (6.99 ± 1.19)%; t = 5.813, P < 0.05], and parasitemia remained at a low level ranging from 0.9% to 1.0% prior to 23 days post-infection. Conclusion A high-throughput screening approach for anti-Babesia drugs has been successfully established, and multiple compounds with high in vitro anti-parasitic activities have been identified. Dacinostat shows favorable efficacy against both B. duncani and B. microti.

Screening and efficacy evaluation of PCNA and NEDD8 as early serological diagnostic antigens for clonorchiasis

LI Yuanyuan, GAO Yuwei, YI Haibo, DU Xinyue, ZHOU Pincheng, WU Chenyun, ZHU Tingjun, GUO Simin, QIAN Menbao, WANG Zhaojun

2026, 44(2):  244-251.  doi:10.12140/j.issn.1000-7423.2026.02.014

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Objective To identify the optimal antigen for early serological diagnosis of clonorchiasis screened from five functionally distinct Clonorchis sinensis candidate proteins. Methods The transcriptome data of C. sinensis were retrieved from Sequence Read Archive in NCBI, together with protein function information, were used to screen five candidate diagnostic antigens, including fructose-1,6-bisphosphate aldolase (FBA), cysteine protease 1 (CP1), proliferating cell nuclear antigen (PCNA), niemann-pick type C2 protein (NPC2), and neural precursor cell expressed developmentally downregulated protein-8 (NEDD8). The expression of antigens was detected in larvae freshly released from C. sinensis metacercariae and adults. Antigen epitopes were predicted using immune epitope databases and spatial epitope prediction tools for protein antigen, and corresponding genes were amplified from C. sinensis cDNA using PCR assay to construct a prokaryotic expression vector pET-302/NT-His. Following induction in Escherichia coli with isopropyl-β-D-thiogalactoside (IPTG), the recombinant protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Serum samples were collected from New Zealand white rabbits and BALB/c mice infected with C. sinensis metacercariae via oral gavage for 6 weeks, early-stage clonorchiasis patients (infected for 2 to 6 weeks), late-stage clonorchiasis patients (infected for more than 6 weeks), healthy controls and individuals with other parasitic infections. Serum specific IgM antibody was detected using indirect enzyme-linked immunosorbent assay (ELISA), with C. sinensis adult antigen as a positive control, and the ratio of absorbance of positive serum to negative serum (P/N) at 450 nm (ΔA₄₅₀ value) served as an evaluation indicator. The sensitivity and specificity were estimated with the software GraphPad Prism 10, and the area under the receiver operating characteristic (ROC) curves (AUC) was estimated. Results Transcriptomic analysis showed that the five candidate proteins were highly expressed in both larvae freshly released from C. sinensis metacercariae and adults. Structural and epitope predictions suggested that antigen epitopes contained multiple linear and conformational B cell epitopes, and their key residues were located in the epitope-exposed region, indicating high antigenic potential. Recombinant expression of five proteins (FBA, CP1, PCNA, NPC2, and NEDD8) was successfully completed, and these five recombinant proteins were successfully purified. SDS-PAGE displayed clear bands, with relative molecular masses consistent with theoretical values. The purities of recombinant proteins were 86.2%, 90.6%, 97.1%, 82.5%, and 93.6%, and the concentrations of recombinant proteins were 1 780, 514, 594, 375, and 1 263 μg/mL, respectively. ELISA measured the strongest IgM response to PCNA and NEDD8, with P/N values (rabbit/mouse) of 1.675/4.176 and 1.521/4.076, respectively. The IgM responses to CP1, FBA and NPC2 were relatively weak, with P/N values (rabbit/mouse) of 1.478/1.870, 0.952/2.531, 1.040/2.413, respectively. The ΔA₄₅₀ values for PCNA (0.304 ± 0.302), NEDD8 (0.506 ± 0.383), and their combinations (0.553 ± 0.358) were all higher in detection of serum samples from early-stage clonorchiasis patients relative to health controls [(0.028 ± 0.017), (0.066 ± 0.044) and (0.066 ± 0.041)] (Z = 3.924, 3.763 and 4.116; all P < 0.01) or late-stage patients [(0.035 ± 0.030), (0.070 ± 0.060) and (0.076 ± 0.071)] (Z = 3.890, 4.116 and 4.386; all P < 0.01). The sensitivities of PCNA, NEDD8 alone and in combinations were 10/14, 11/14, and 12/14 for diagnosis of serum samples from early-stage clonorchiasis patients, and the specificity was all 95.5% (21/22). ROC curve analysis showed that the AUC of PCNA, NEDD8 alone and in combinations were 0.873 4, 0.879 9, and 0.915 6, respectively. Conclusion PCNA and NEDD8 may be used as diagnostic antigens for detection of serum IgM antibody from individuals with early-stage C. sinensis infections. PCNA-NEDD8T combinations may improve the early diagnostic performance, which has potential for early immunodiagnosis of clonorchiasis.

Genetic evolutionary characteristics of and prevalence of parasitic infections in tabanid flies in Shiqu County, eastern Qinghai-Tibet Plateau

LI Mengqing, WANG Xu, YANG Yang, XUE Chuizhao, ZUO Qingqiu, YIN Jianhai, CAO Jianping

2026, 44(2):  252-259.  doi:10.12140/j.issn.1000-7423.2026.02.015

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Objective To investigate the distribution characteristics of and prevalence of parasitic infections in tabanid flies in Shiqu County, eastern Qinghai-Tibet Plateau, so as to provide baseline data for understanding tabanid fly distribution patterns and formulation of tabanid-borne disease control strategies in this region. Methods Tabanid samples were collected from Nixia Town, Sexu Town, and Luoxu Town of Shiqu County, Sichuan Province using a net trapping method from 10:00 to 14:00 in August 2023 and August 2025. Genomic DNA was extracted from tabanid samples, and target fragments were amplified and sequenced using 28S rRNA-specific primers. Sequences were assembled using the Geneious Prime software, and the species was identified with the BLAST tool in the GenBank database. Haplotype diversity was calculated using the DnaSP software, and saturation of base substitutions was tested with the DAMBE software. Nucleotide substitution models were screened with the jModelTest tool, and a phylogenetic tree was generated with the maximum likelihood method using the MEGA software. High-throughput sequencing was performed with universal parasite primers, and sequencing data were subjected to quality control, paired-end assembly, operational taxonomic unit (OTU) clustering to detect potential parasites in tabanid samples. Results A total of 324 tabanid specimens were sampled, and 304 valid samples were obtained following identification and screening, which belonged to three genera and three species, including Hybomitra bimaculate (Tabanidae), Tabanus rufofrator (Tabanidae), and Symphoromyia hirta (Rhagionidae). T. rufofrator was the dominant species (275 individuals, 90.4%, 275/304), and 24 (7.89%, 24/304) H. bimaculate and 5 S. hirta (1.65%, 5/304) were identified. Totally eight haplotypes were detected, including two for H. bimaculata and three each for T. rufofrator and S. hirta. Phylogenetic analysis revealed clear differentiation structures of the study sequences at family, genus, and species levels. Two H. bimaculata haplotypes (H3 and H4) were clustered into the same clade with the reference sequence with the GenBank accession number of M243434, and three T. rufofrator haplotypes (H1, H6 and H8) were clustered into the monophyletic clade with the reference sequence with the GenBank accession number of AF238561, while three S. hirta haplotypes (H2, H5 and H7) and the reference sequence with the GenBank accession number of AF238558 generated a highly supported monophyletic clade. High-throughput sequencing yielded 1 499 OUT, which contained two potential parasite-associated OUT, including Mastophorus muris and Romanomermis iyengari, which were only present in H. bimaculate, while no parasites were detected in T. rufofrator or S. hirta. Conclusion T. rufofrator is the dominant species of the tabanid fly community in Shiqu County, with an overall low genetic diversity. Low-abundance nematode sequences are detected in H. bimaculate, suggesting the potential ecological significance in the transmission of tabanid-borne pathogens on the Qinghai-Tibet Plateau.

Preparation and assessment of qualitative reference materials for detection of Cryptosporidium parvum nucleic acid

WU Longfei, XU Huiyan, YU Changhong, CHEN Xinyi, LI Junqiang, ZHANG Sumei, ZHANG Longxian

2026, 44(2):  260-265.  doi:10.12140/j.issn.1000-7423.2026.02.016

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Objective To prepare reference materials for detection of Cryptosporidium parvum nucleic acid according to procedures and processes for preparation of reference materials specified in the national criteria GB/T 15000 Directives for the work of reference materials. Methods A large number of oocysts of the C. parvum C1HN strain (subtype IIdA19G1) were obtained through calf passage, and purified, and genomic DNA was extracted from C. parvum oocysts using DNA extraction kits. Then, the source of oocyst DNA was traced. Once the traceability of the extracted DNA was verified, DNA samples were aliquoted and cryopreserved. The homogeneity, freeze-thaw stability, and short- and long-term stability of C. parvum oocyst DNA samples were evaluated with nested PCR and quantitative Real-time PCR (qPCR) assays, and the constant values were determined collaboratively by multiple institutions. All statistical analyses were performed using the software SPSS 27.0, and differences of means were tested for statistical significance with one-way analysis of variance (ANOVA) and paired-samples t test. Results The PCR amplification fragment was approximately 830 bp in size, which was consistent with the results detected by the national standard. Sequencing revealed that the SSU rRNA gene fragments on chromosomes 2, 7, and 8 of this strain exhibited 99.65% sequence identity with those of the C1HN strain, indicating complete concordance with the infecting strain, and the subtype was identified as ⅡdA19G1. Homogeneity tests showed that the Ct value of the samples was 15.00 ± 0.36, with a mean square of 0.17 between groups and 0.11 within groups, indicating no statistically significant difference (F = 1.49, P > 0.05). Freeze-thaw stability tests showed that the Ct values of the samples were 15.67 ± 0.25, 15.68 ± 0.22, 15.69 ± 0.27, 15.71 ± 0.27 on days 0 (t = 0.00, P > 0.05), 3 (t = 0.31, P > 0.05), 7 (t = 0.50, P > 0.05), and 14 following repeated freeze-thaw cycles (t = 0.71, P > 0.05), respectively. Short-term stability tests showed that the Ct values were 16.48 ± 0.28, 16.48 ± 0.25, 16.49 ± 0.28, 16.50 ± 0.25, 16.53 ± 0.26, 16.56 ± 0.25, 16.57 ± 0.24 following storage at 4 ℃ for 0 (t = 0, P > 0.05), 1 (t = 0.04, P > 0.05), 2 (t = 0.34, P > 0.05), 3 (t = 0.46, P > 0.05), 4 (t = 1.56, P > 0.05), 5 (t = 1.60, P > 0.05), and 10 days (t = 1.84, P > 0.05); 16.20 ± 0.18, 16.21 ± 0.14, 16.21 ± 0.13, 16.22 ± 0.14, 16.23 ± 0.14, 16.24 ± 0.14, and 16.25 ± 0.15 following storage at 25 ℃ for 0 (t = 0.00, P > 0.05), 1 (t = 0.17, P > 0.05), 2 (t = 0.28, P > 0.05), 3 (t = 0.47, P > 0.05), 4 (t = 0.61, P > 0.05), 5 (t = 1.09, P > 0.05), and 10 days (t = 1.40, P > 0.05); and 16.21 ± 0.17, 16.22 ± 0.16, 16.23 ± 0.16, 16.24 ± 0.17, 16.26 ± 0.18, 16.25 ± 0.18 and 16.26 ± 0.19 following storage at 37 ℃ for 0 (t = 0.00, P > 0.05), 1 (t = 0.33, P > 0.05), 2 (t = 0.56, P > 0.05), 3 (t = 1.10, P > 0.05), 4 (t = 1.20, P > 0.05), 5 (t = 1.34, P > 0.05), and 10 days (t = 1.89, P > 0.05), respectively. Long-term stability tests showed that the Ct values were 15.67 ± 0.11, 15.67 ± 0.21, 15.67 ± 0.21, 15.67 ± 0.25, 16.67 ± 0.24 and 15.67 ± 0.23 following storage at -20 ℃ for 0 (t = 0.00, P > 0.05), 30 (t = 0.01, P > 0.05), 90 (t = 0.11, P > 0.05), 180 (t = 0.23, P > 0.05), 270 (t = 0.33, P > 0.05), and 360 days (t = 0.39, P > 0.05), respectively. Nested PCR assays of samples stored for 180 and 360 days amplified specific bands with approximately 830 bp in size, and sequencing results were consistent with those previously obtained. All participating institutions provided verification reports confirming that the prepared reference materials might be used as a qualitative positive control for detection of C. parvum nucleic acid. Conclusion The prepared reference material meets the requirements specified in the national standard GB/T 15000 Directives for the work of reference materials with reliable traceability, good homogeneity, stability in compliance with specifications, and accurate constant values.

Combined treatment of deworming and thoracoscopic surgery for refractory pediatric paragonimiasis-induced encapsulated pleural effusions: a single-center experience of 15 cases and discussion on surgical protocols

WANG Jie, GONG Sheng, GAO Yi

2026, 44(2):  266-270.  doi:10.12140/j.issn.1000-7423.2026.02.017

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Objective To evaluate the clinical efficacy, surgical timing, and therapeutic value of deworming with praziquantel in combination with thoracoscopic surgery for treatment of pediatric refractory paragonimiasis-induced encapsulated pleural effusions. Methods The medical records of 15 children with paragonimiasis-induced encapsulated pleural effusions who underwent surgical treatment in Public Health Clinical Center of Chengdu from January 2015 to January 2025 were retrospectively analyzed. All cases received initial treatment with praziquantel pre-surgery at a dose of 75 mg/(kg·d), divided into 3 oral doses, for 3 consecutive days as one course of treatment, with an interval of one month between each course. Then, routine single port thoracoscopic surgery was performed a group of thoracic surgeons. Postoperative antibiotics administration, deworming, and rehabilitation training were given, and follow-up re-examinations were performed at 1, 3, 6, and 12 months post-surgery. Results The 15 children included 11 males and 4 females, with a median age of 8.50 (5.46, 10.79) years, and all cases had a thoracic-pulmonary type of paragonimiasis, including 7 cases complicated by abdominal type, 3 cases complicated by pericardial type, one case complicated by cerebral type, and one case complicated by subcutaneous nodule type. There were 14 children tested positive for serum IgG antibodies against Paragonimus, and the other one case was definitively diagnosed with paragonimiasis by pleural pathology despite sero-negativity. Preoperative chest CT scans displayed encapsulated effusions with pleural thickening and adhesions among all children, and 2 to 6 courses of treatment with praziquantel were given. All cases successfully completed thoracoscopic surgery, and typical empyema and fibrous septa were observed during the operation. Charcot-Leyden crystals were found in postoperative pathological specimens from 15 children, including parasite fragments seen in 2 cases and parasite eggs in one case. Routine blood tests at one-month post-surgical follow-up revealed that the absolute value of peripheral blood eosinophils in decreased from 3.70 (1.32, 8.33) × 10⁹/L pre-surgery to 0.34 (0.23, 0.56) × 10⁹/L post-surgery among all 15 children (Z = -3.41, P < 0.01), and eosinophils percentage decreased from 33.00% (16.60%, 44.70%) pre-surgery to 4.20% (2.10%, 6.50%) post-surgery (Z = -3.19, P < 0.01). All symptoms were relieved, and imaging showed good lung recruitment and no serious complications. Conclusion Combined treatment of deworming and thoracoscopic surgery may effectively remove lesions, promote lung recruitment and remarkably reverse host eosinophilic inflammation among children with paragonimiasis-induced encapsulated pleural effusions and unsatisfactory efficacy of medical treatment.

Epidemiological characteristics and laboratory testing of Plasmodium falciparum malaria in Hubei Province from 2020 to 2024

YI Jia, LIN Wen, WU Dongni, DONG Xiaorong, SUN Lingcong, ZHU Hong, ZHANG Cong, TU Zhen, XIA Jing

2026, 44(2):  271-276.  doi:10.12140/j.issn.1000-7423.2026.02.018

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Objective To investigate the epidemiological characteristics, clinical manifestations and laboratory tests of Plasmodium falciparum malaria in Hubei Province from 2020 to 2024, so as to provide insights into prevention of development of severe and fatal cases. Methods Data pertaining to P. falciparum malaria cases reported in Hubei Province from 2020 to 2024 were collected from the National Notifiable Disease Reporting System and the National Parasitic Disease Control Information Management System. The temporal, spatial and population distributions, healthcare-seeking behaviors and laboratory testing of P. falciparum malaria cases were statistically analyzed. Results A total of 251 P. falciparum malaria cases were reported in Hubei Province from 2020 to 2024, including 250 imported cases (99.60%, 250/251) and one case caused by blood transfusion, and 31 severe cases (12.35%, 31/251) and 2 deaths (0.80%). P. falciparum malaria consisted 68.02% (251/369) of all malaria cases, and the proportion of P. falciparum malaria cases in all malaria cases varied in years (χ² = 35.431, P < 0.01). The majority of imported P. falciparum malaria cases acquired infections from Africa (98.80%, 247/250), with the Democratic Republic of Congo (65 cases), Nigeria (46 cases), and Guinea (19 cases) as three largest sources of importation. The largest number of reported P. falciparum malaria cases was found in January, with males aged 31 to 50 years as the predominant demographic. Wuhan City (86 cases), Huangshi City (41 cases), and Yichang City (33 cases) as the three most highly affected cities, and P. falciparum malaria cases were reported in 70.87% (73/103) of counties (districts). The proportion of definitive diagnosis of P. falciparum malaria cases was74.90% (188/251) at initial diagnosis, and lower proportions were observed at village clinics (1/13) and among private practitioners (1/8). The proportions of definitive diagnosis of P. falciparum malaria cases were 73.08% (19/26), 89.22% (91/102), 81.93% (68/83), and 25.00% (10/40) at provincial-, city-, county-, and township and lower-level medical institutions, respectively (χ² = 66.325, P < 0.01). The median interval from disease onset to healthcare seeking was 1 day among P. falciparum malaria cases, with 54.18% (136/251) seeking healthcare services within 1 day of onset and 81.67% (205/251) within 3 days, and the median interval from healthcare seeking to definitive diagnosis was 1 day, with 65.74% (165/251) confirmed within 1 day of seeking healthcare services and 88.45% (222/251) within 3 days. Fever was the most frequent symptom (98.41%, 247/251), and other common symptoms included chills (57.37%, 144/251), sweating (42.23%, 106/251), headache (47.41%, 119/251), and myalgia (10.36%, 26/251). Complications occurred in 16.33% (41/251) of subjects, including severe anemia and liver and kidney injury. The detection rates of P. falciparum malaria were 98.33% (236/240), 95.42% (229/240), and 100% (240/240) in 240 anticoagulant blood samples from P. falciparum cases by rapid diagnostic tests (RDTs), microscopy, and quantitative Real-time PCR assay, respectively (χ² = 12.664, P < 0.05). P. falciparum was identified in peripheral blood smears from 240 P. falciparum malaria cases, with ring-stage alone seen in 70.42% (169/240) of blood smears, rings and trophozoites seen in 20.83% (50/240), and parasites at all developmental stages (rings, trophozoites, schizonts and gametocytes) in 5.00% (12/240). There were 34.17% (82/240) of cases with parasitemias ranging from 1/μL to 100 000/μL, and 21.25% (51/240) with parasitemias of > 250 000/μL. Multivariate logistic regression analysis identified parasitemia was an independent risk factor for severe malaria (OR > 1, P < 0.05). Conclusion A large number of P. falciparum malaria cases with widespread distributions were reported in Hubei Province from 2020 to 2024, and most cases acquired infections in Africa. Fever was the major clinical symptom. Some cases experienced a prolonged duration for a definitive diagnosis, and the proportion of severe case was high among imported P. falciparum malaria cases. Future efforts should focus on improving patients’ awareness of seeking healthcare services, shortening the duration to definitive diagnosis, providing timely and standardized treatment, in order to reduce the development of severe and fatal cases.

Economic burden of hospitalized patients with echinococcosis in Xinjiang from 2014 to 2023

WANG Le, GU Ruijuan, TONG Chao, NIU Jiangshui, A Mengke, SU Xiaohong, REN Yanjun, LIU Jingdong, DENG Dawei, WEN Hao

2026, 44(2):  277-283.  doi:10.12140/j.issn.1000-7423.2026.02.019

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Objective To estimate the overall economic burden of echinococcosis patients in Xinjiang from 2014 to 2023 and to identify its influencing factors, so as to provide the scientific evidence for formulation and improvements of medical control strategies for echinococcosis in Xinjiang. Methods A total of 8 hospitals were sampled from Xinjiang by means of stratified sampling and convenience sampling, including 4 Class Ⅲ Grade A hospitals and 4 Class Ⅱ Grade A hospitals. Medical records were collected from hospitalized patients with a primary diagnosis of echinococcosis from January 1, 2014 to December 31, 2023, and variables were retrieved from medical records, including gender, age, ethnicity, length of hospital stay, hospitalization cost, hospital level, region, surgical treatment, health insurance status, and type of echinococcosis. Subjects’ demographics and cost distribution were analyzed with descriptive statistics. Univariate analysis was performed with Mann-Whitney U test and Kruskal-Wallis H test, and independent factors affecting hospitalization costs were identified using a logarithmic multiple linear regression model (stepwise method). Indirect economic burden was calculated with disability-adjusted life year (DALY) combined with the human capital method using the following formula: indirect economic burden = gross national product per capita × DALY × productivity weight. SPSS 24.0 was used for statistical analyses. Results A total of 5 184 hospitalized patients with echinococcosis were included. The number of patients discharging from hospital peaked in 2017 (865 cases), dropped to the lowest in 2022 (230 cases, a decrease of 73.4% compared with the peak), and rebounded to 361 cases in 2023. The subjects had a median age of 44 years, and were primarily at ages of 45 to 59 years (32.9%, 1 703 cases). Of all subjects, 54.5% (2 823 cases) were men, and 90.1% (4 670 cases) suffered from cystic echinococcosis. Hospitalization costs showed a skewed distribution, with the median increasing from 23 137.2 Yuan in 2014 to 34 921.0 Yuan in 2022. The mean hospitalization cost was higher for alveolar echinococcosis patients (71 302.7 Yuan) than for cystic echinococcosis patients (29 397.3 Yuan; Z = -11.750, P < 0.05), and the mean hospitalization cost was higher among patients from northern Xinjiang (36 283.3 Yuan) was higher than those from southern Xinjiang (17 703.7 Yuan)(Z = -19.701, P < 0.05). The highest mean cost was seen among patients with length of hospital stay exceeding 30 days (106 761.4 Yuan; H = 1 263.033, P < 0.05). Logarithmic multiple linear regression analysis identified length of hospital stay (β = 0.414), surgical treatment (β = ‒0.471), hospital level (β = 0.210), region, type of echinococcosis, age, and health insurance status as independent factors affecting hospitalization costs (adjusted R² = 0.553, P < 0.05). Indirect economic burden analysis showed that echinococcosis caused a total of 10 007.3 DALYs lost during the study period, with an mean loss of 2.0 DALYs per patient. Patients at ages of 15 to 44 years had the highest disease burden, with a total DALY loss of 5 465.0 person-years, accounting for 54.6% of the total loss. The total indirect economic burden was 423.823 million Yuan, with a mean indirect economic burden of 82 000 Yuan per patient. Conclusion The economic burden of echinococcosis patients shows significant structural differences in Xinjiang. Alveolar echinococcosis is the core disease type leading to high medical costs, and its resource consumption pattern is essentially different from that of cystic echinococcosis. Young and middle-aged populations at ages of 15 to 44 years have more than half of the productivity loss and is the main population affected by indirect economic burdens.

Factors affecting human alveolar echinococcosis in Baqing County, Xizang Autonomous Region: a case control study

Danzeng Quzhen, Bianba Zhuoma, Ci Duojie, Suolang Lamu, Suolang Wangjie, Kangzhu Yixi, Laba Sangzhu, Gongsang Quzhen

2026, 44(2):  284-289.  doi:10.12140/j.issn.1000-7423.2026.02.020

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Objective To identify the main influencing factors of human alveolar echinococcosis (AE) in Baqing County, Xizang Autonomous Region, so as to provide insights into echinococcosis control in pastoral areas. Methods AE patients that were definitively diagnosed with abdominal B-mode ultrasonography and based on WS 257-2006 Diagnostic Criteria for Echinococcosis in the Human Echinococcosis Screening Programme in Baqing County, Xizang Autonomous Region from January 2023 to October 2024 were selected as the case group, and residents that were free of echinococcosis as revealed by abdominal B-mode ultrasonography during the same time served as controls. Matching was performed based on the same gender, similar age (± 5 years), and residence in the same or geographically adjacent administrative villages, with one case matched to two controls. A face to face questionnaire survey was conducted by uniformly trained investigators, and the questionnaire covered general demographics, dog breeding and management, environmental exposure factors, and personal behavioral habits. All statistical analyses were performed using the software R, with chi square test used for between group comparisons, and a conditional logistic regression model for identifying factors affecting the risk of AE. Results A total of 258 residents from Baqing County were enrolled, including 86 cases in the case groups and 172 matched controls, and all subjects were Tibetan residents. The mean age was (44.6 ± 14.2) years in the case group and (44.1 ± 14.1) years in the control group, with no statistically significant difference （t = 0.26, P > 0.05）, and no significant differences were observed in gender, occupation, educational level, body mass index, marital status, or household size (χ² = 0.00, 1.53, 0.83, 4.09, 0.71 and 0.53, all P > 0.05). Univariate conditional logistic regression analysis identified significant associations of dog breeding (OR = 2.82, 95% CI: 1.45-5.48), never deworming for dogs (OR = 3.22, 95% CI: 1.09-9.48), improper disposal of dog feces (OR = 4.86, 95% CI: 2.09-11.30), frequent contact with dogs (OR = 2.98, 95% CI: 1.42-6.24), presence of foxes around the residence (OR = 2.42, 95% CI: 1.37-4.27), use of river/stream water as the main drinking water source (OR = 2.55, 95% CI: 1.26-5.16), drinking unboiled water (OR = 2.02, 95% CI: 1.17-3.48), and frequent consumption of unwashed fresh Cordyceps (OR = 3.87, 95% CI: 1.55-9.67) with the risk of AE (all P < 0.05), and multivariate analysis identified dog breeding duration for > 5 years (OR = 4.68, 95% CI: 1.09-20.17), non disposal of dog feces (OR = 9.35, 95% CI: 2.39-36.64), frequent contact with dogs (OR = 6.92, 95% CI: 1.21-39.51), and drinking water sourced from rivers, lakes, ditches, or ponds (OR = 4.97, 95% CI: 1.07-23.20) as main risk factors for AE. Conclusion The development of human AE is closely associated with dog management and drinking water safety in Baqing County. It is recommended to reinforce dog management and standardized disposal of dog feces, improve drinking water sanitation, and enhance health education among herders.

REVIEWS

Nanoparticles and their application in vaccine research for important parasitic diseases

WU Keke, WANG Liqun, LUO Xuenong

2026, 44(2):  290-295.  doi:10.12140/j.issn.1000-7423.2026.02.021

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Parasitic diseases remain a major global public health concern. Conventional chemotherapy frequently faces limitations, including significant adverse effects and the inability to prevent reinfection. Although prophylactic vaccination represents an ideal strategy for achieving long-term control of parasitic diseases, the development of traditional vaccines has been severely impeded by the complex life cycles, high antigenic variability, and sophisticated immune evasion mechanisms of parasites. In recent years, nanoparticles-leveraging their unique physicochemical properties to integrate antigen protection, targeted delivery, and synergistic immune activation-have provided a new opportunity to overcome the bottlenecks in parasitic vaccine development. In light of this, this article systematically reviews the physicochemical characteristics of various nanoparticles and their latest advances in vaccine research against major parasitic diseases, such as schistosomiasis, toxoplasmosis, and echinococcosis. It provides an in-depth discussion on their mechanisms of action and core advantages in optimizing antigen delivery and immunomodulation. Furthermore, it objectively analyzes the current challenges and future trends facing nanovaccines in clinical translation and industrial application, aiming to provide theoretical insights for the rational design and development strategies of novel parasitic vaccines.

Advances in neurological injury and sequelae of cerebral malaria

CUI Weiwei, CHENG Yang

2026, 44(2):  296-304.  doi:10.12140/j.issn.1000-7423.2026.02.022

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Cerebral malaria is one of the most fatal complications of Plasmodium falciparum infection, characterised clinically by altered consciousness or coma. Asexual P. falciparum parasites can be detected in peripheral blood, after exclusion of other causes of encephalopathy. The mortality rate of this disease can reach up to 20%, with approximately 15%-25% of survivors experiencing varying degrees of long-term neurological dysfunction, significantly impairing quality of life and social functioning. Current research has identified the retention of infected red blood cell in cerebral microvasculature, neuroinflammatory cascades, and blood-brain barrier disruption as core pathogenic mechanisms; however, the molecular regulatory networks underlying these processes remain incompletely elucidated. In this article, we systematically review recent research progress on the pathogenesis and neurological sequelae of cerebral malaria, focusing on key links in central nervous system damage, with the aim of providing a theoretical basis for the development of targeted adjuvant therapies and interventions.

CASE REPORT

Diagnostic and therapeutic analysis of a case of cysticercosis involving the nervous system and subcutaneous muscles

ZHANG Yan, WEN Yang, ZHU Yu

2026, 44(2):  305-307.  doi:10.12140/j.issn.1000-7423.2026.02.023

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A 8-year-old male Tibetan child was admitted to the West China Second Hospital of Sichuan University on October 25, 2024, due to intermittent fever, headache and vomiting. The patient had long resided in Mangkang County, Changdu City, Xizang Autonomous Region, with cattle and sheep raised in his family, and he had a history of consuming raw beef and mutton. Physical examination revealed a palpable mass near the elbow joint on the medial side of the left upper arm, measuring 2 cm × 1.5 cm with no significant tenderness. Serological tests showed positive IgG antibodies against Echinococcus, and liver function tests indicated alanine aminotransferase at 59 U/L and aspartate aminotransferase at 58 U/L. Respiratory multiplex pathogen nucleic acid assay tested positive for rhinovirus. Tapeworm eggs were identified in stool samples on October 29, October 30, and November 6. Head MRI scans (plain scan + contrast enhancement) displayed multiple space-occupying lesions in the bilateral cerebral hemispheres, presenting as circle-like long T1 and long T2 signal shadows, and superficial ultrasound of the left upper arm showed a cystic-solid mass within the muscular layer of biceps brachii. On November 1, 2024, a subcutaneous and intramuscular nodule resection was performed in the left upper arm, and pathological biopsy identified parasites in the muscle and fibrous tissues, which confirmed the diagnosis of neurocysticercosis, subcutaneous, and muscular cysticercosis. The child was given anti-parasitic treatment with albendazole [15 mg/(kg·d)] and praziquantel [(50 mg/(kg·d)] for 10 days, followed by anti-inflammatory therapy with dexamethasone. He had improvements and was then discharged from hospital. After discontinuation for 3 days, drug administration was resumed for another 10 days. Follow-up calls at 3 and 6 months post-discharge showed no significant abnormalities.