Table of Content

30 August 2025, Volume 43 Issue 4

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SPECIAL REPORT

National surveillance of soil-transmitted helminth infecions in China in 2022

ZHU Huihui, HUANG Jilei, ZHOU Changhai, ZHU Tingjun, ZHAO Luyuan, QIAN Menbao, LI Shizhu

2025, 43(4):  451-457.  doi:10.12140/j.issn.1000-7423.2025.04.001

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Objective To investigate the current status of and trends in prevalence of soil-transmitted helminth (STH) infections in China, so as to provide insights into management of soil-transmitted helminthiasi. Methods In 2022, a nationwide surveillance of soil-transmitted helminthiasis was conducted at designated surveillance sites of soil-transmitted helminthiasis across 30 provinces (autonomous regions/municipalities) in China (this survey was not conducted in Xizang), and a unified sampling method was employed in each surveillance site. One administrative village was selected from eastern, western, southern, northern and central regions, respectively. Two hundred permanent residents at ages of 3 years and older were sampled using a cluster sampling method from each administrative village, with a total of 1 000 individuals in a surveillance site. Participants’ stool samples were collected and tested for STH infections and egg counts using the modified Kato-Katz thick smear method (two slides for one sample). The prevalence and intensity of STH infections were calculated, and differences of prevalence were tested for statistical significance with chi-square test. Five households were randomly selected from each administrative village, and one soil sample was collected from each household farmland/vegetable garden for isolation and characterization of hookworm larvae and detection of Ascaris lumbricoides eggs. Results A total of 363 603 residents from 354 surveillance sites in 30 provinces (autonomous regions/municipalities) were surveyed for STH infections, with an overall infection prevalence of 0.64% (2 330/363 603), with the highest prevalence detected in Sichuan Province (4.53%, 776/17 149), followed by in Yunnan Province (4.31%, 646/14 974) and Hainan Province (3.98%, 123/3 089). Fifteen provinces (autonomous regions/municipalities), including Fujian, Guizhou, Jiangxi, Zhejiang, Chongqing, Shandong, Qinghai, Guangxi, Ningxia, Anhui, Hunan, Gansu, Shanxi, Liaoning, and Guangdong, showed prevalence ranging from 0.10% to 1.50%. Seven provinces (autonomous regions), namely Jilin, Shaanxi, Xinjiang, Jiangsu, Henan, Hubei, and Hebei, exhibited prevalence between > 0 and < 0.10%. No infected cases were detected in five provinces (autonomous regions/municipalities): Beijing, Heilongjiang, Inner Mongolia, Shanghai, and Tianjin. The prevalence of STH infections was higher among females (0.72%, 1 332/184 152) than among males (0.56%, 998/179 451) (χ² = 39.89, P < 0.05). The highest prevalence of STH infections was detected among residents at ages of 60 years and older (1.00%, 1 014/100 963). The differences in prevalence among age groups were statistically significant (χ² = 366.01, P < 0.05). The prevalence rates of hookworm, A. lumbricoides, and Trichuris trichiura were 0.39% (1 410/363 603), 0.16% (569/363 603), and 0.14% (494/363 603), with mild infections accounting for 94.47% (1 332/1 410), 71.00% (404/569), 91.50% (452/494), respectively. Both A. lumbricoides eggs and hookworm larvae were detected in soil samples from farmlands and vegetable gardens. The detection rates of A. lumbricoides eggs and hookworm larvae were 1.24% (29/2 335) and 1.11% (26/2 335), respectively. Conclusion The overall prevalence of soil-transmitted helminthiasis is low in China; however, there are high regional and human disparities. Tailored prevention and control measures should be implemented to promote transmission control and interruption of soil-transmitted helminthiasis in China.

CONSENSUS AND GUIDELINE

Technical measures for joint prevention and control of imported malaria in the Yangtze River Delta: Experts consensus from Shanghai, Jiangsu, Zhejiang and Anhui

ZHU Min, ZHU Guoding, WANG Xiaoxiao, XU Xian, WANG Zhenyu, CAO Yuanyuan, FENG Yan, ZHANG Tao, ZHANG Chengang, ZHANG Xuan, YU Qing

2025, 43(4):  458-462.  doi:10.12140/j.issn.1000-7423.2025.04.002

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China was certificated malaria-free by WHO in 2021, however, the global malaria epidemic remains severe. Imported malaria cases frequently occur in the Yangtze River Delta, a core region with active economy and frequent international exchanges in China, where the risk of re-transmission remains persistently. To continuously consolidate malaria elimination achievements and implement the Outline of Regional Integrated Development Plan for the Yangtze River Delta, a consensus was reached on technical measures for joint prevention and control of imported malaria in the Yangtze River Delta by malaria control professionals from Shanghai, Jiangsu, Zhejiang and Anhui centers for disease control and prevention (institute of parasitic diseases), including opening up channels for information sharing and collaboration within the region, technical coordination for monitoring and early warning techniques among population at a high risk of malaria, technical coordination and intensification of joint response techniques for malaria epidemics, coordinative development of reserves for malaria surveillance, early warning and prevention and control skills, coordinative guarantee for reserve and requisition of antimalarial drugs and coordinative development of health science popularization standards and precision in the region, following full discussions and adequate preparation. In addition, future cooperation needs and prospects have been proposed based on the Public Health Integration Platform in the Yangtze River Delta, so as to further improve and optimize regional joint prevention and control techniques to effectively respond to current challenges in trans-regional prevention and control of imported malaria.

EXPERT VIEWPOINT

Health threat and ecological function of parasites

WANG Xu, YIN Jianhai, HAN Shuai, YANG Yang, LI Mengqing, SHEN Yujuan, CAO Jianping

2025, 43(4):  463-468.  doi:10.12140/j.issn.1000-7423.2025.04.003

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Along with the ubiquitous parasitic relationship, parasites are widely distributed across all ecological systems in the world. The parasitic behavior of parasites to the host may cause multiple parasitic diseases, posing persistent threats to human and animal health. Meanwhile, as key participants in functional networks of ecosystems, parasites play irreplaceable ecological roles in maintaining the earth’s ecosystem homeostasis through regulating host population dynamics, energy flow and biodiversity. This review analyzes the dual biological role of parasites from perspectives of pathogenic threats and ecological services, and discusses the potential application of interdisciplinary approaches in parasitology and parasitic diseases research under the collaborative framework of “One Health” and “Ecohealth”.

ORIGINAL ARTICLES

Effect of splenic egg granulomas on the structure and function of mouse splenic lymphoid follicles at a late stage of Schistosoma japonicum infections

WANG Yanjuan, ZHANG Yanjun, ZHOU Xiaojun, CAO Jianping

2025, 43(4):  469-474.  doi:10.12140/j.issn.1000-7423.2025.04.004

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Objective To examine the effect of mouse splenic granulomas on the structure and function of splenic lymphoid follicles at a late stage of Schistosoma japonicum infectio. Methods Fifteen C57BL/6 mice were infected with 20 S. japonicum cercariaes each via abdominal skin. Mice were sacrificed 16 weeks post-infection for spleen sampling, and divided into two groups presence of egg granulomas on the spleen surface, including the splenic granuloma group and non-granuloma group, while mice 5 weeks post-infection (8 mice) and healthy mice (7 mice) served as infection and healthy controls. The spleens of mice in each group were weighed and splenic lymphocytes were collected, and the structure of mouse splenic lymphoid follicles was observed using HE staining. The distribution of positive mucosal addressin cell adhesion molecule-1 (MAdCAM-1⁺) cells was observed in the marginal zone of lymphoid follicles using indirect immunofluorescence, and the distribution of Ki67⁺ and PNA⁺ germinal center cells was observed in mouse spleens using immunohistochemical and direct immunofluorescence. Mouse splenic CD4⁺ T cells were isolated with magnetic beads, and the proportion of follicular helper T (Tfh) cells in mouse splenic CD4⁺ T cells was detected using flow cytometry. Results More severe splenomegaly was seen in mice infected with S. japonicum for 16 weeks in relative to mice infected with S. japonicum for 5 weeks and healthy mice, and the mousse spleen weight was significantly lower in the splenic granuloma group (0.37 ± 0.04) than in the non-granuloma group (0.48 ± 0.04) (F = 266.17, P < 0.01). HE and indirect immunofluorescence revealed a decrease in the number of splenic lymphoid follicles, follicle areas, counts of MAdCAM-1⁺ cells in the marginal zone in the splenic granuloma group, while no lymphoid follicles or their marginal zone structures were detected in the non-granuloma group. Immunohistochemical and direct immunofluorescence demonstrated decreased numbers of Ki67⁺ and PNA⁺ germinal center cells in the splenic granuloma group compared with healthy controls, while no Ki67⁺ or PNA⁺ germinal center cells were observed in the non-granuloma group. Flow cytometry detected that the proportion of positive inducible costimulator (ICOS⁺) and chemokine C-X-C motif receptor 5 (CXCR5⁺) Tfh cells was significantly higher in mouse splenic CD4⁺ T cells in the splenic granuloma group [(0.54 ± 0.06)%] than in the non-granuloma group [(0.36 ± 0.05)%] (F = 61.68, P < 0.01）, and the proportion of ICOS⁺ positive programmed cell death protein 1 (PD1⁺) Tfh cells was significantly higher in mouse splenic CD4⁺ T cells in the splenic granuloma group [(5.25 ± 0.17)%] than in the non-granuloma group [(2.40 ± 0.09)%] (F = 29.13, P < 0.01), and the proportion of ICOS⁺CXCR5⁺PD1⁺ Tfh cells was higher in the splenic granuloma group [(0.42 ± 0.06)%] than in the non-granulomas group [(0.26 ± 0.05)%] (F = 20.03, P < 0.01）. Conclusion Alleviated splenic pathological damages and more intact internal functional structures that generate antibodies, such as germinal center and follicles, are seen in mice with splenic granulomas than in those without granulomas at a late stage of S. japonicum infection, which may result in a higher antibody level in mice with splenic granulomas than in those without granulomas at a late stage of S. japonicum infection.

Screening of marker of hepatic stellate cells apoptosis in schistosomiasis japonica-induced liver fibrosis

DONG Bowen, ZHONG Haoran, ZHU Danlin, LI Hao, LU Ke, FU Zhiqiang, LIU Jinming, JIN Yamei

2025, 43(4):  475-481.  doi:10.12140/j.issn.1000-7423.2025.04.005

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Objective To screen molecular markers suitable for labeling host hepatic stellate cell (HSC) apoptosis in schistosomiasis japonica-induced liver fibrosis (SSLF). Methods Amino acid sequences of apoptosis-related proteins from Mus musculus and Schistosoma japonicum were obtained through literature review and database retrieval, and homology analysis of these proteins was performed using the Protein BLAST tool on the NCBI website. Liver tissues were collected from mice infected with S. japonicum 4, 8 and 12 weeks post-infection, and uninfected mice served as controls. Liver tissues were sectioned, and the expression of HSC marker Desmin and apoptosis-related molecules was detected using immunofluorescence staining. The dynamics of mouse HSC apoptosis were analyzed at different time points post-infection to identify molecular markers indicative of host cell apoptosis in SSLF. In addition, sequence homology and phylogenetic analysis of apoptosis-related proteins sequences from different adaptive hosts of S. japonicum were performed using the software Jalview and MEGA11. All data were analyzed with one-way analysis of variance (ANOVA) using the software GraphPad Prism 8. Results Sixteen S. japonicum apoptosis-related proteins and eighteen M. musculus apoptosis-related proteins were identified through literature review and database retrieval. Sequence similarity analysis showed 20% to 80% homology between sequences of M. musculus and S. japonicum apoptosis-related proteins, and no homologous proteins of M. musculus Fas or Fas ligand (FasL) were found in S. japonicum. Immunofluorescence staining showed remarkable distribution of apoptosis-related proteins (green fluorescence) Cleaved caspase 3, B cell-lymphoma-2 associated death promoter (Bad), and FasL surrounding egg granulomas in mice 8 weeks post-infection. Notably, Cleaved caspase 3 and Bad bound specifically to both mouse liver tissues and schistosome eggs, while FasL exclusively labeled host liver tissues, and was co-localized significantly with Desmin (red fluorescence). More importantly, the intensity of FasL and Desmin co-localization increased over time points post-infection. The proportion of FasL⁺Desmin⁺ cells (apoptotic HSCs) in Desmin⁺ cells (HSCs) was comparable between infected (0) and uninfected mice (0) 4 weeks post-infection (P > 0.05), and the proportions were 35.36% and 89.12% in infected mice 8 and 12 weeks post-infection, which were both significantly higher than in the uninfected group (t = 13.38, 28.77; both P < 0.01). Sequence alignment analysis showed that the homology of M. musculus FasL sequence was 91.04% with Rattus norvegicus, 80.57% with Microtus fortis, 75.09% with Homo sapiens, 76.24% with Sus scrofa, 76.34% with Ovis aries, 77.99% with Bubalus bubalis, 75.27% with Equus caballus, 74.48% with Canis lupus familiaris, and 77.86% with Felis catus. Phylogenetic analysis indicated that M. musculus FasL sequence was more closely related to R. norvegicus than to M. fortis, H. sapiens, S. scrofa, O. aries, B. bubalis, E. caballus, C. lupus familiaris, and F. catus. Conclusion FasL may be feasible tolabel host HSCs apoptosis in schistosomiasis japonica-induced liver fibrosis.

Endemic situation of schistosomiasis in national surveillance sites of Anhui Province from 2020 to 2024

XU Xiaojuan, DING Songjun, DAI Bo, LIU Ting, GAO Fenghua, ZHANG Shiqing

2025, 43(4):  482-488.  doi:10.12140/j.issn.1000-7423.2025.04.006

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Objective To investigate the changes in the endemic situation of schistosomiasis in national surveillance sites of Anhui Province, so as to provide the scientific evidence for promoting the schistosomiasis elimination strategy in the provinc. Methods According to the requirements of the National Schistosomiasis Surveillance Scheme in China (2020 Edition), national schistosomiasis surveillance were assigned in 50 endemic counties (cities, districts) across Anhui Province. During the period between October and November from 2020 to 2024, schistosomiasis surveillance was performed among permanent residents at ages of over 6 years in surveillance villages from class I surveillance counties, with no less than 300 residents in each village, and schistosomiasis screening was conducted among mobile populations by means of active surveillance and sentinel hospitals-based passive surveillance, with no less than 200 populations in each county. Serological screening of schistosomiasis was performed using indirect hemagglutination test, and sero-positive individuals were subjected to parasitological examinations using egg hatching tests with nylon gauzes (three slides for one sample) and modified Kato-Katz thick smear technique (three slides for one sample). During the period between October and December from 2020 to 2024, Schistosoma japonicum infection was detected in livestock using the miracidium hatching test. Oncomelania hupensis snail distribution was surveyed using systematic sampling combined with environmental sampling in each spring from 2020 to 2024, and S. japonicum infection was detected in O. hupensis snails using microscopy. S. japonicum nucleic acid was detected in O. hupensis snails using loop-mediated isothermal amplification (LAMP) assay in class Ⅰ and Ⅱ surveillance counties where the transmission of schistosomiasis was achieved during the recent 5 years, and the surveillance of wild feces and snail distribution was performed in snail-infested habitats in some administrative villages in class Ⅰ surveillance counties and some class Ⅱ surveillance counties in each spring from 2020 to 2024. Results Serological screening for schistosomiasis was conducted among local residents at 63 868 person-times in Anhui Province from 2020 to 2024, and there were 908 sero-positives detected (530 males and 378 females). The seroprevalence of schistosomiasis reduced from 1.49% (263/17 644) in 2020 to 1.23% (118/9 592) among local residents in 2024. Serological screening for schistosomiasis was conducted among mobile populations at 55 587 person-times in Anhui Province from 2020 to 2024, with 104 sero-positive cases detected (71 males and 33 females), with a reduction from 0.41% (43/10 596) in 2020 to 0.07% (8/10 889) in 2024. Nevertheless, no egg-positives were identified among local residents or mobile populations. A total of 5 812 livestock were tested for S. japonicum infections, with no infections identified. A total of 7 810 settings were surveyed for snail distribution from 2020 to 2024, covering an area of 593.85 km², and the area with snail habitats reduced from 37.80 km² in 2020 to 34.90 km² in 2024. A total of 1 101 819 frames were surveyed for snails using a systematic sampling method, and the 5-year mean occurrence of frames with snails and density of living snails were 8.51% and 0.30 snails per 0.11 m², respectively. The highest occurrence of frames with snails was seen in ditches (12.16%, 8 174/672 091), dry lands (6.57%, 489/2 952), ditches (13.55%, 7 455/55 019), ditches (10.13%, 5 198/51 316), and ditches (10.19%, 6 669/65 441) from 2020 to 2024, respectively, and there were significant differences in the occurrence of frames with snails in different types of endemic areas from 2020 to 2024 (χ² = 2 012.52, 3 174.12, 5 501.59, 1 454.46, 720.04; all P < 0.01). The occurrence rates of frames with snails were higher in the hilly endemic areas (11.32%, 10.55%, 10.19%, 10.68%, and 9.48%) than in marshland and lake endemic areas (7.72%, 9.60%, 7.91%, 7.17%, and 7.63%) from 2020 to 2024 (χ² = 740.27, 35.61, 216.66, 433.64, 151.03; all P < 0.01). No S. japonicum infections were identified in snails during the intermediate host monitoring or risk monitoring, and S. japonicum infection was detected in only one wild bovine stool sample in 2022, with no S. japonicum infections identified in other wild stool samples. Conclusion The prevalence of schistosomiasis appeared an overall tendency towards a decline in Anhui Province from 2020 to 2024; however, there was still a risk of schistosomiasis transmission. Improved and intensified construction of the schistosomiasis surveillance system is recommended to provide insights into implementation of the precise schistosomiasis control strategy.

Evaluation of implementation quality of schistosomiasis surveillance programmes in surveillance provinces of China in 2024

GUO Suying, LI Yinlong, LI Shizhen, DANG Hui, ZHANG Lijuan, HE Junyi, YANG Fan, ZHU Hongqing, JIA Tiewu, QIN Zhiqiang, FENG Ting, DENG Wangping, LV Chao, YANG Ying, HAO Yuwan, SUN Junling, CAO Chunli, XU Jing, LI Shizhu

2025, 43(4):  489-496.  doi:10.12140/j.issn.1000-7423.2025.04.007

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Objective To understand and evaluate the implementation quality and problems of current schistosomiasis surveillance programmes in China, so as to provide insights into facilitating schistosomiasis surveillance programme. Methods In October to December, 2024, a total of 22 counties (county-level cities or districts, hereinafter referred to as “counties”) were sampled using a quota sampling method from 13 provinces (autonomous regions, municipalities, hereinafter referred to as “provinces”) where national schistosomiasis surveillance programmes were implemented. Based on Information technology―Evaluation indicators for data quality (GB/T 36344-2018) and the expert meeting method, evaluation indicators were proposed to assess the quality of the surveillance programmes, including standardization, integrity, accuracy, consistency, and timeliness. A total of 50 subjects’ serum samples, 30 Kato-Katz slides (all slides were examined if less than 30 slides collected from residents were available), and 10 snail survey environments were sampled from each survey county for re-reviews. The indicator scores were analyzed using the equal weighting method, and the average scores and the coincidence rates of serum and etiologic re-review samples were calculated. In addition, the difference in the average score was compared with rank sum test between elimination, nonendemic areas and non-elimination areas. Results The 22 evaluation counties included 7 surveillance counties and 2 nonendemic counties in schistosomiasis elimination provinces, and 13 epidemic surveillance counties in non-elimination provinces. The average score was (9.04 ± 0.79) points in 22 evaluation counties, and (8.92 ± 0.76) points in surveillance counties in non-elimination provinces and (9.22 ± 0.83) points in surveillance and nonendemic counties in elimination provinces (Z = -0.889, P > 0.05). The average scores for standardization, integrity, accuracy, consistency, and timeliness of schistosomiasis surveillance programmes were (1.86 ± 0.35), (1.73 ± 0.46), (1.77 ± 0.43), (1.95 ± 0.21), and (1.73 ± 0.46) points in 22 survey counties, respectively. Among the survey counties, there were 3 counties with insufficient surveillance funds, 6 with incomplete original records of snail surveys, 3 with inconsistent qualitative between original and re-review results of 7 serum samples, 3 with inaccurate snail monitoring results (including longitude and latitude of snail surveillance, and snail survey diagram), 1 with inconsistent original data with total numbers in statistical tables, and 6 with failure in timely data update, entry, and management. Conclusion The quality of national schistosomiasis surveillance programmes is high in surveillance counties in China; however, the timeliness and standardization remain to be further improved. Sustainable fund and material supports and improved capability through training among monitoring professionals are required to ensure the quality of surveillance programmes.

Selection and identification of aptamers against the circulating cathodic antigen of Schistosoma mansoni

TANG Qi, QIU Jiayin, LI Jiajia, ZHOU Xinjie, LV Chao, FENG Ting, XU Jing, QIN Zhiqiang

2025, 43(4):  497-502.  doi:10.12140/j.issn.1000-7423.2025.04.008

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Objective To screen and identify the aptamer specifically targeting the circulating cathodic antigen (CCA) of Schistosoma mansoni. Methods The recombinant prokaryotic expression vector pET-28a-CCA was constructed, and the expression of pET-28a-CCA protein was induced and purified by Ni-NTA column. The expression characteristics of pET-28a-CCA were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using pET-28a-CCA protein as the target, systematic evolution of ligands by exponential enrichment (SELEX) was performed. Quantitative real-time PCR was used to monitor and analyze each round of library enrichment. After multiple rounds of stringent selection, the obtained products were subjected to high-throughput sequencing analysis, bioinformatics analysis, and the characterization of binding affinity of candidate aptamers to the pET-28a-CCA protein using microscale thermophoresis (MST), followed by molecular docking. Results Double enzyme digestion and sequencing results confirmed that the pET-28a-CCA recombinant plasmid contained an approximately 1 044 bp CCA gene fragment. SDS-PAGE results demonstrated successful expression of the pET-28a-CCA protein, with a relative molecular mass (M_r) of approximately 43 600. The SELEX screening results showed that the library of the 13th screening round reached the best enrichment. Based on sequencing results, nine candidate aptamer sequences were selected. Among them, seven aptamers exhibited binding affinity to the pET-28a-CCA as determined by MST analysis. TQ-apt1 had the lowest Gibbs free energy (ΔG = -13.09 kcal/mol), the highest GC content (59.2%), the highest melting temperature (102.3 ℃), and the highest G-quadruplex score (15 points). TQ-apt1 had the highest affinity with pET-28a-CCA protein with a dissociation constant of 2.1 nmol/L. Conclusion In this study, aptamers targeting the pET-28a-CCA protein were successfully obtained through SELEX technology.

Interaction between IFITM3 and Foxp3 in mice during Echinococcus multilocularis infections

YUAN Zhen, DIDAER Yeergeming, WANG Fei, JIANG Tao, DUAN Mingjun, AERZIGULI Tuerxun, QI Xinwei, SHAN Jiaoyu

2025, 43(4):  503-510.  doi:10.12140/j.issn.1000-7423.2025.04.009

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Objective To investigate the interaction between interferon-inducible transmembrane protein 3 (IFITM3) and forkhead box P3 (Foxp3) in mice during Echinococcus multilocularis infections. Methods E. multilocularis protoscolices were obtained from gerbil liver tissues, and a C57BL/6J mouse model of E. multilocularis infection was established via intraperitoneal injection with E. multilocularis protoscolices. Mice were euthanized 15, 30, 60 and 90 days post-infection, and mouse liver tissues were collected, with uninfected mice as controls. Pathological changes of mouse liver tissues were observed at different time points using hematoxylin-eosin (HE) staining, Masson staining and immunohistochemistry, and the relative expression of IFITM3 and Foxp3 proteins was determined in liver tissues using Western blotting. HEK-293T cells were transfected with IFITM3-ShRNA interference plasmids (ShIFITM3-1, ShIFITM3-2, ShIFITM3-3), and the optimal knockdown fragments were screened for dose-dependent experiments (transfected at 0, 0.5, 1.0, 1.5, 2.0 μg) to further detect changes in Foxp3 expression. The IFITM3-Foxp3 molecular docking was analyzed using the AutoDock software, and the tag plasmid HA-FITTM3 and Flag-Foxp3a were transfected into HEK-293T cells. Total cellular proteins were extracted for immunoprecipitation to verify the interaction between FITTM3 and Foxp3a. Results HE staining revealed disordered arrangements of liver tissues and gradually aggravated infiltration of inflammatory cells in liver tissues of mice infected with E. multilocularis. Masson staining displayed no significant blue staining areas in the control group, and a gradual increase in the blue staining area in the infection group, with (22.12 ± 3.09)%, (28.57 ± 2.1)%, (41.20 ± 2.01)%, and (58.30 ± 2.21)% proportions 15, 30, 60, and 90 days post-infection, respectively (F = 135.60, P < 0.01). Immunohistochemical staining showed that the positive expression rate of IFITM3 increased from (20.18 ± 7.25)% in the control group to (56.22 ± 4.49)% at 90 days post-infection (F = 25.40, P < 0.05), and the positive expression rate of Foxp3 increased from (2.12 ± 1.49)% in the control group to (59.10 ± 4.45)% at 90 days post-infection (F = 106.30, P < 0.05). Western blotting assay showed that the relative expression level of IFITM3 decreased to 0.27 ± 0.04 at 15 days post-infection, and then increased and reached 1.01 ± 0.05 at 90 days post-infection (F = 52.37, P < 0.05), and the relative expression level of Foxp3 gradually increased over time, from 0.03 ± 0.01 in the control group to 0.97 ± 0.03 at 90 days post-infection (F = 143.20, P < 0.05). The IFITM3 expression positively correlated with Foxp3 expression (R² = 0.687 3, P < 0.05). Transfection with ShIFITM3-1, ShIFITM3-2, and ShIFITM3-3 plasmids all resulted in a decrease in the relative expression level of IFITM3 (0.32 ± 0.02, 0.23 ± 0.05, 0.53 ± 0.09), with ShIFITM3-2 showing the highest knockdown efficiency (F = 37.05, P < 0.01), and the relative expression of Foxp3 gradually decreased with ShIFITM3-2 transfection at 0 (1.10 ± 0.14), 0.5 (0.62 ± 0.05), 1.0 (0.52 ± 0.04), 1.5 (0.49 ± 0.01), and 2.0 μg (0.33 ± 0.05), respectively (F = 42.06, P < 0.01). Molecular docking showed that the docking score between IFITM3 and Foxp3 was -312 kcal/mol, indicating a good affinity, and immunoprecipitation indicated an interaction between IFITM3 and Foxp3. Conclusion There is an interaction between IFITM3 and Foxp3 in mice during E. multilocularis infections, and IFITM3 and Foxp3 expression is positively correlated with the severity of liver damages.

Genetic structure characteristics of Anopheles sinensis populations in different regions of Anhui Province

WANG Shuqi, JIANG Jingjing, LV Xiaofeng, CHU Qinshu, XU Xian, LU Xuechun, LIU Zijian, ZHANG Tao, YIN Jianhai

2025, 43(4):  511-517.  doi:10.12140/j.issn.1000-7423.2025.04.010

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Objective To investigate the genetic diversity, genetic differentiation and phylogenetic relationships of Anopheles sinensis populations in different ecological regions of Anhui Provinc. Methods Adult An. mosquitoes were captured from Huangshan District, Nanling County, Feidong County, Dingyuan County, Suixi County and Guzhen County that were randomly selected from three ecological regions of north of the Yangtze River, areas between the Yangtze River and Huaihe River and north of the Huaihe River from 2023 to 2024. Following morphological identification, genomic DNA was extracted from mosquitoes, and the ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunitⅠ(COⅠ) genes were amplified using PCR assay. Following bidirectional sequencing of the amplified products, the yielded sequences were subjected to BLAST alignment in the National Center for Biotechnology Information (NCBI). The obtained sequences were aligned using the software MEGA 11. The inter-population genetic distance was calculated, and phylogenetic trees were built. The polymorphism information content of An. sinensis populations was calculated using the software DnaSP 5 and mismatch analysis was performed. Analysis of molecular variance (AMOVA) and neutrality tests were performed using the software Arlequin 3.5.2.2, and the genetic differentiation (F_ST) and gene flow (Nm) values were calculated. In addition, the SPSS 18.0.0 software was used to generate scatter plots and examine the correlation between inter-population geographical distance and genetic distance. Results A total of 300 An. sinensis samples were obtained. After PCR amplification of the rDNA-ITS2 sequence, a specific band appeared at approximately 490 bp in length, which was consistent with the size of An. sinensis (GenBank accession number: MG816544.1). Following PCR amplification of the COⅠsequence, BLAST alignment showed that the sequence had a 99% identity with that of An. sinensis. The A + T content (68.8%) was higher than the G + C content (31.1%) in the COⅠgene sequence, showing a remarkable AT bias. A total of 129 polymorphic sites and 204 haplotypes were identified, including 166 unique haplotypes and 38 shared haplotypes. The haplotypes Hap_11, Hap_12, Hap_15, Hap_26, and Hap_38 were detected in the COⅠgene sequences of mosquitoes from all three ecological regions, with Hap_26 shared by the most populations and present in all six counties (districts). The haplotype diversity was 0.994, and the nucleotide diversity was 0.010 1. Among the six counties (districts), Nanling County had the highest values in terms of numbers of polymorphic sites and haplotypes, haplotype diversity, and nucleotide diversity. The results of AMOVA showed that the intra-population variation of An. sinensis was 97.5% in six counties (districts), and the inter-population variation was 2.5% (P < 0.01). The F_ST values of An. sinensis were all < 0.1 in six counties (districts), and the Nm values were all > 1. The Tajima’s D values of An. sinensis populations were all negative in six counties (districts), with P values of < 0.05 for Tajima’s D values in Guzhen County, Huangshan District, and Nanling County, and the Fu’s Fu test also supported the results of Tajima’s D test. The mismatch distribution of An. sinensis populations all showed an obvious multimodal structure in six counties (districts). In addition, there was no correlation between the genetic distance and geographical distance of An. sinensis collected from six counties (districts) (r² = 0.109, P > 0.05). Phylogenetic analysis revealed no geographical clusters of An. sinensis collected from the six counties (districts). Conclusion There are high genetic polymorphisms, sufficient intra-population gene flow and no remarkable genetic differentiation in An. sinensis populations in different ecological regions of Anhui Province; however, population expansion has been identified recently.

Rapid detection of mosquito species and their transmitted pathogens based on the CRISPR/Cas12a system

LI Jianyong, HE Biao, LI Meilin, LIU Taiping, ZHU Feng, ZHANG Jian, XU Wenyue

2025, 43(4):  518-525.  doi:10.12140/j.issn.1000-7423.2025.04.011

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Objective To develop an assay for rapid detection of mosquito species and their transmitted pathogens based on the clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) system, so as to improve the surveillance and control efficiency of mosquito-borne disease. Methods Genomic DNA was extracted from 30 first-instar Aedes albopictus larvae and 10 one-day-old adult Anopheles stephensi. Species-specific genomic sequences were amplified using recombinase polymerase amplification (RPA). Ae. albopictus genomic DNA was subjected to RPA with specific RPA primers of An. stephensi to assess the specificity of RPA. The CRISPR/Cas12a system was employed for fluorescence detection, and An. stephensi and Ae. albopictus-derived CRISPR RNA (crRNA) was employed for cross-reactivity evaluation to assess the specificity of the CRISPR/Cas12a system. Following recovery of cryopreserved Plasmodium yoelii, three mice were intravenously injected with 1 × 10⁶ P. yoelii each via tail vein. On day 3 post-infection, 100 An. stephensi mosquitoes were allowed to engorge on infected mice. Mosquitoes were dissected 1, 2, 3, 4, 5 and 10 days following blood meals and their midguts were sampled and observed for Plasmodium oocysts under a microscope. An. stephensi mosquitoes were infected with Plasmodium, and genomic DNA was extracted from mosquito pools 1, 2, 3, 4, 5 and 10 days post-infection. Plasmodium 18S rRNA gene fragment was amplified using RPA and CRISPR/Cas12a-based fluorescence detection. CRISPR/Cas12a-based fluorescence detection was performed with Plasmodium genomic DNA at serial dilutions of 10^-9, 10^-10, 10^-11, 10^-12, 10^-13, 10^-14, 10^-15, 10^-16, 10^-17 and 10^-18 mol/L to evaluate the lowest limit of detection of the system. Dengue virus serotypes Ⅰ, Ⅱ, ⅢandⅣ-specific sequences were synthesized, and cloned into the pUC18 plasmid vector. The nonstructural protein 5 (NS5) gene fragment was amplified using RPA, and then detected using CRISPR/Cas12a-based fluorescence assay. Dengue virus serotypesⅠ, Ⅱ, Ⅲ and Ⅳ cRNA were mixed with other dengue virus plasmids to evaluate the specificity of the CRISPR/Cas12a system, and CRISPR/Cas12a-based fluorescence detection was performed with Plasmodium genomic DNA at serial dilutions of 10^-9, 10^-10, 10^-11, 10^-12, 10^-13, 10^-14, 10^-15, 10^-16, 10^-17, 10^-18 and 10^-19 mol/L to evaluate the lowest limit of detection of the system. In addition, Cas12-based specific nucleic acid test strips were employed for visualized detection of An. stephensi, Ae. albopictus, Plasmodium, and dengue virus. Results RPA assay yielded the An. stephensi cytochrome c oxidase subunit 1 (cox1) gene fragment with 89 bp in length and Ae. albopictus cox1 gene fragment with 87 bp in length. CRISPR/Cas12a system detected robust activation of LbCas12a trans-cleavage activity by both Ae. albopictus and An. stephensi cox1 gene fragments, with detectable fluorescence signals emerging within 5 minutes of reaction initiation. The maximal signal intensity was observed for adult An. stephensi samples, reaching equilibrium by 13 minutes. RPA assay of P. yoelii 18S rRNA gene produced a fragment with 142 bp in length. CRISPR/Cas12a detection confirmed P. yoelii infection in An. stephensi samples 1, 2, 3, 4, 5, 10 days post-infection, with the lowest limit of detection of 3.3 × 10^-17 mol/L. Microscopic examinations revealed no oocyst formation on days 1 to 4 following blood meals, two observable oocysts on day 5, and progressive increased in both oocyst burdens and sizes on day 10. RPA assay of dengue virus serotypesⅠ, Ⅱ, Ⅲ and Ⅳ NS5 gene fragments generated serotype-specific products of 109, 93, 147, and 170 bp, respectively. CRISPR/Cas12a system detected dengue virus sequences in samples with detectable fluorescence signals within 5 minutes and peak intensity attained by 17 minutes. In addition, no inter-serotypic cross-reactivity was observed, with the lowest limit of detection of 2.5 × 10^-18 mol/L, and the Cas12-based nucleic acid test strips achieved visualized detection of target virus sequences within 30 minutes. Conclusion A rapid, highly sensitive and specific, visualized CRISPR/Cas12a-based assay has been established for identification of mosquito species and detection of pathogens, which provides an effective tool for early warning and transmission interruption of mosquito-borne diseases.

Trend analysis and forecast of global burden of malaria from 1990 to 2035

YANG Guobing, HE Aiwei, QIN Yu, YANG Jian, WANG Jichun

2025, 43(4):  526-532.  doi:10.12140/j.issn.1000-7423.2025.04.012

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Objective To analyze the trends in global burden of malaria from 1990 to 2021 and to predict the trends in malaria burden from 2022 to 2035, so as to provide a scientific basis for the global malaria control programm. Methods Demographic data, and the incidence, prevalence, mortality, and disability-adjusted life years (DALY) rates of malaria were extracted from the Global Burden of Disease 2021 Database, and the age-standardized incidence rate (ASIR), age-standardized prevalence rate (ASPR), age-standardized mortality rate (ASMR), age-standardized DALY rate (ASDR) of malaria with their 95% uncertainty intervals (UI), and socio-demographic index (SDI) were extracted. The trends in ASIR, ASPR, ASMR and ASDR of malaria from 1990 to 2021 were examined using average annual percent change (AAPC) with 95% confidence interval (CI). The global burden due to malaria was projected from 2022 to 2035 with a Bayesian age-period-cohort (BAPC) model, and the trends in ASIR, ASPR, ASMR and ASDR of malaria from 2022 to 2035 were analyzed with AAPC and 95%CI. Results Significant declines were observed globally in ASIR [AAPC = -7.25%, 95%CI: (-11.68%, -2.81%)], ASPR [AAPC = -14.42%, 95%CI: (-15.99%, -12.84%)], ASMR [AAPC = -0.06%, 95%CI: (-0.08%, -0.05%), and ASDR [AAPC = -4.86%, 95%CI: (-5.81%, -3.91%)] of malaria from 1990 to 2021. Across different SDI regions, the lowest SDI region demonstrated the most substantial decline in all standardized indicators, with AAPCs of -184.95% [95%CI: (-193.26%, -176.64%)], -253.13% [95%CI: (-256.83%, -249.43%)], -0.75% [95%CI: (-0.82%, -0.68%)], and -65.59% [95%CI: (-69.07%, -62.11%)], respectively. In 2021, the Central sub-Saharan Africa region had the highest malaria ASIR [21 152.56 per 100 000, 95%UI: (16 857.61, 26 956.39) per 100 000] and ASPR [21 328.79 per 100 000, 95%UI: (16 092.29, 28 557.44) per 100 000], whereas Western sub-Saharan Africa had the highest ASMR [106.48 per 100 000, 95%UI: (38.23, 222.04) per 100 000] and ASDR [5 668.41 per 100 000, 95%UI: (2 216.18, 11 127.48) per 100 000]. At the national level, Liberia reported the highest ASIR [27 702.66 per 100 000, 95%UI: (14 565.45, 38 887.49) per 100 000] and ASPR [29 248.89 per 100 000, 95%UI: (13 845.02, 47 808.36) per 100 000]; Burkina Faso had the highest ASMR [173.13 per 100 000, 95%UI: (75.38, 318.49) per 100 000]; and Sierra Leone recorded the highest ASDR [8 940.31 per 100 000, 95%UI: (3 029.75, 17 358.47) per 100 000]. Among different age groups, children under 5 years had the highest number of incident cases (96.303 4 million) and prevalent cases (31.749 million), as well as the highest incidence [14 631.95 per 100 000, 95%UI: (10 175.30, 21 120.91) per 100 000] and prevalence [4 823.86 per 100 000, 95%UI: (4 362.74, 5 361.59) per 100 000]. Globally, malaria ASIR, ASPR, ASMR, and ASDR showed strong inverse correlations with SDI (r = -0.89, -0.89, -0.87, -0.87, respectively; all P < 0.01). It was projected with the BAPC model that declining trends were seen in ASIR [AAPC = -17.12%, 95%CI: (-17.24%, -16.99%)], ASPR [AAPC = -5.93%, 95%CI: (-6.07%, -5.80%)], ASMR [AAPC = -0.10%, 95%CI: (-0.10%, -0.11%)], and ASDR [AAPC = -9.63%, 95%CI: (-9.65%, -9.62%)] of malaria from 2022 to 2035. Conclusion The global burden of malaria declined continuously from 1990 to 2021, with children under five years of age identified as high-risk populations. Forecasts suggest an overall tendency towards a continued decline in the global burden of malaria from 2022 to 2035.

Epidemiological characteristics of imported malaria in Zhejiang Province from 2020 to 2024

ZHANG Xuan, WANG Xiaoxiao, CHEN Hualiang, ZHANG Jiaqi, XU Wenjie, LU Qiaoyi, FENG Yan, RUAN Wei

2025, 43(4):  533-539.  doi:10.12140/j.issn.1000-7423.2025.04.013

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Objective To investigate the epidemiological characteristics of imported malaria cases in Zhejiang Province, so as to provide insights into management of imported malari. Methods All data pertaining to malaria cases reported in Zhejiang Province from 2020 to 2024 were collected from China Disease Prevention and Control Information System and Parasitic Diseases Prevention and Control Information System, and malaria parasite species, temporal, spatial and population distributions, source of infections, time interval from illness onset to initial diagnosis, time interval from initial diagnosis to definite diagnosis, and relapse or recrudescence were descriptively analyzed among reported malaria cases. Chi-square test and nonparametric tests were used to compare differences. Results A total of 448 malaria cases were reported in Zhejiang Province from 2020 to 2024, with annual numbers of 53, 44, 64, 118 and 169 cases, respectively, and all cases were imported cases. Falciparum malaria was the predominant type, accounting for 77.23% (346/448), while ovale malaria, vivax malaria and malariae malaria accounted for 13.39% (60/448), 6.03% (27/448), and 3.35% (15/448), respectively. The reported malaria cases acquired infections across four continents, with Africa as the main source of infection (95.54%, 428/448) and there were 3 cases with cross-border mosquito-borne malaria. Malaria cases were reported across all 11 prefecture­level cities in Zhejiang Province, with Hangzhou (29.24%, 131/448), Jinhua (25.67%, 115/448), and Wenzhou (10.49%, 47/448) reporting the highest number of cases. The number of reported malaria cases peaked in August in 2022 (15 cases) and remained elevated from July to October in both 2023 and 2024 (11-21 cases monthly). The reported cases included 412 males and 36 females, with a male-to-female ratio of 11.4 ∶ 1, and had a median age of 44 years. The accuracy of initial diagnosis was 75.89% (340/448) among reported malaria cases, and the median time intervals from illness onset to initial diagnosis and from initial diagnosis to definite diagnosis were both one day. 59.38% (266/448) of reported malaria cases sought healthcare within one day of illness onset, while 5.58% (25/448) sought treatment after more than 5 days. The proportion of falciparum malaria cases seeking healthcare within 2 days of onset (78.03%, 270/346) was higher than that of non-falciparum malaria cases (65.69%, 67/102) (χ² = 6.445, P < 0.05). There were 72.32% (324/448) of malaria cases receiving definite diagnosis within one day of initial diagnosis, while 4.91% (22/448) were diagnosed more than 5 days. The proportion of falciparum malaria cases diagnosed within 2 days (85.84%, 297/346) was higher than that of non-falciparum malaria cases (76.47%, 78/102) (χ² = 5.068, P < 0.05). In addition, total of 8 cases with malaria relapse or recrudescence were reported in Zhejiang Province, including 4 cases with falciparum malaria, 3 cases with ovale malaria and one case with vivax malaria. Conclusion The number of imported malaria cases appeared an upward trend in Zhejiang Province from 2020 to 2024. Intensified surveillance-response interventions and improved malaria diagnostic capability are recommended to prevent re-establishment of imported malaria.

Endemic status of amoebic dysentery in China from 2020 to 2021

HUANG Jilei, ZHOU Changhai, ZHU Tingjun, ZHU Huihui, LI Shizhu, QIAN Menbao

2025, 43(4):  540-546.  doi:10.12140/j.issn.1000-7423.2025.04.014

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Objective To investigate the incidence, epidemiological characteristics and changing trends of amoebic dysentery in China, so as to provide insights into formulation of tailored control strategies and surveillance measures and optimization of resource allocation in the futur. Methods Individual data and demographic data of cases of amoebic dysentery with dates of disease onset between January 1, 2020 and December 31, 2021 were collected from the Infectious Disease Report Information Management System, and the incidence and its changing trends of amoebic dysentery were analyzed using a descriptive epidemiological method in China from 2020 to 2021. In addition, the spatial distribution characteristics and tempo-spatial clusters of amoebic dysentery were identified using the ArcGIS and SaTScan software. Results A total of 670 and 539 cases of amoebic dysentery were reported in China in 2020 and 2021, with incidence of 0.048/100 000 and 0.038/100 000, respectively. The reported incident cases and incidence of amoebic dysentery in 2021 decreased by 19.55% and 20.83% as compared with those in 2020, respectively. The number of reported incident cases peaked in May 2020 (85 cases) and June 2021 (73 cases), and the period from May to July was the common epidemic season for both years. The ratios of male to female reported incident cases were 1.27 ∶ 1 and 1.28 ∶ 1 in 2020 and 2021, respectively. Both the reported incident cases and incidence of amoebic dysentery appeared a tendency towards a decline followed by a rise, and then a decline with ages, and the highest numbers of reported incident cases and incidence of amoebic dysentery were all found at ages of 0 to 5 years, with 326 incident cases and 0.40/100 000 incidence in 2020 and 197 incident cases and 0.25/100 000 incidence in 2021. The reported incident cases were predominantly found among diaspora children in 2020 and 2021, accounting for 45.82% (307/670) and 33.77% (182/539) of totally reported cases, followed by farmers, accounting for 22.09% (148/670) and 26.53% (143/539), respectively. The provinces (autonomous regions) with the three highest incidence were Guangxi (0.32/100 000), Henan (0.16/100 000) and Guangdong (0.12/100 000) in 2020, and Guangxi (0.22/100 000), Yunnan (0.19/100 000) and Guangdong (0.09/100 000) in 2021, respectively. Both Guangxi and Guangdong were among the top three provinces (autonomous regions) in both years in terms of both reported incidence and incident cases of amoebic dysentery, which both appeared a tendency towards a decline from 2020 to 2021. Global spatial autocorrelation analysis revealed no significant spatial clustering of amoebic dysentery at either a provincial or county level. Space-time scan analysis detected two statistically significant clusters in 2021, and the cluster areas were found in Yunnan and Guizhou, which involved 17 counties (cities, districts) in southwestern China. Conclusion The incident cases and incidence of amebiasis appeared a tendency towards a decline in China from 2020 to 2021, with two cluster areas in southwestern China.

Clinical and epidemiologic features of visceral leishmaniasis: A retrospective analysis of 49 cases

YAN Dongning, ZHANG Keru, LI Changling, CHEN Xiaojun

2025, 43(4):  547-554.  doi:10.12140/j.issn.1000-7423.2025.04.015

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Objective To investigate the epidemiological and clinical characteristics and prognosis of visceral leishmaniasis (VL) patients in Gansu Province, so as to provide references for clinicians in the diagnosis and treatment of VL among clinician. Methods The epidemiological data, clinical manifestations, laboratory tests and prognosis of VL patients admitted to Gansu Provincial Hospital during the period between May 2011 and October 2024 were retrospectively analyzed. All statistical analyses were conducted using the software SPSS 26.0. Intergroup comparisons of categorical variables were tested for statistical significance with χ² test, while continuous variables conforming to a normal distribution were compared with independent samples t-test. Results A total of 49 VL patients were admitted to Gansu Provincial Hospital from May 2011 to October 2024, including 34 men (69.4%) and 15 female (30.6%). The patients were aged from 1 to 73 years, including 17 (34.7%) minors (age < 18 years) and 32 (65.3%) adults (age of 18 years and older). In terms of geographical locations, 49.0% (24/49) resided in Longnan City, 30.6% (15/49) in Gannan Tibetan Autonomous Prefecture, and 20.4% (10/49) in other non-endemic areas of Gansu Province. All 49 patients presented with fever and chills, predominantly demonstrating an irregular fever pattern, and splenomegaly was observed in 87.8% (43/49), hepatomegaly in 22.4% (11/49), beening in 6.1% (3/49). Comorbid complications included respiratory tract infections (44.9%, 21/49), hemophagocytic lymphohistiocytosis (HLH) (24.5%, 12/49), and abnormal liver function was noted in 14.3% (7/49). Hematological abnormalities revealed leukopenia in 81.6% (40/49), decreased hemoglobin levels in 85.7% (42/49), and thrombocytopenia in 67.3% (33/49). Among those with thrombocytopenia, 6 cases exhibited severe reduction in platelet counts (< 20 × 10⁹/L). Serum ferritin levels were quantified among 27 patients, with 96.3% (26/27) showing elevated concentrations exceeding 204 ng/L. All patients underwent biochemical tests, including aspartate aminotransferase (AST), alanine aminotransferase (ALT) and globulin measurements, and elevated AST levels were detected in 55.1% (27/49), increased ALT in 40.8% (20/49), and hyperglobulinemia in 34.7% (17/49). Among 44 patients undergoing serological testing using rK39 immunochromatographic assay, 90.9% (40/44) were tested positive, and bone marrow aspiration was performed among 43 patients, with Leishmania amastigotes identified in 83.7% (36/43) of bone marrow smears. Among these positive cases, 15 exhibited hemophagocytic syndrome. A significantly lower proportion of minor patients (6/17) experienced an interval of exceeding 30 days from disease onset to definite diagnosis as compared to adult patients (22/32, 68.7%) (χ² = 5.074, P < 0.05). Hypertriglyceridemia was observed more frequently among minors (15/17) than among adults (15/30, 50.0%) (χ² = 6.871, P < 0.05), and hemophagocysis in bone marrow was less common among minor patients (3/17) compared to adults (14/30, 46.7%) (χ² = 3.958, P < 0.05). Among the 49 patients, 37 were diagnosed with uncomplicated VL, while 12 presented with HLH-associated VL. The mean platelet count was (46.83 ± 25.54) × 10⁹/L among HLH-associated VL patients, which was significantly lower than among uncomplicated VL patients [(103.14 ± 65.61) × 10⁹/L] (t = 4.309, P < 0.05). Conversely, the mean serum ferritin level was significantly higher among HLH-associated VL cases [(4 440.18 ± 1 140.88) ng/ml] than among uncomplicated VL cases [(1 483.35 ± 1 551.36) ng/ml] (t = -5.509, P < 0.05). Among 46 patients who received treatment at Gansu Provincial Hospital, 29 were treatment naive and 17 had received prior therapies, and all these patients were administered intravenously with sodium stibogluconate (SSG), including one death after 14 days post-treatment due to hemophagocytic syndrome and respiratory failure. Two patients required alternative therapeutic regimens: one was switched to amphotericin B cholesteryl sulfate complex due to adverse effects (nausea, vomiting, abdominal pain and diarrhea) during intravenous infusion of SSG, and another was switched to the same alternative regimen due to persistent detection of Leishmania amastigotes in bone marrow smears after four treatment cycles. All surviving patients showed clinical improvements compared to admission status. Conclusions For patients from VL-endemic areas presenting with fever, hepatosplenomegaly, and bicytopenia or pancytopenia on routine blood tests, prompt serological testing (rK39 immunochromatographic assay) or bone marrow aspiration should be performed. Upon diagnosis as VL, standardized treatment with antileishmanial agents and anti-inflammatory medications should be initiated in a timely manner to improve patients’ prognosis.

Expression and immunoprotective effect of a Toxoplasma gondii four-antigen fusion protein

WU Qinli, NI Ze, DING Haojie, DING Jianzu, ZHENG Bin, ZHUO Xunhui, LU Shaohong

2025, 43(4):  555-561.  doi:10.12140/j.issn.1000-7423.2025.04.016

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Objective To investigate the immunoprotective effect of a four-antigen fusion protein vaccine (referred to as 4 ×) composed of Toxoplasma gondii rhoptry protein 18 (ROP18), centrin family protein TGME49_237490, oocyst wall-specific protein ortholog TGME49_268230, and microneme protein 13 (MIC13). Methods The 4 × target gene was amplified using PCR assay, and recombinant plasmids were constructed and characterized. The successfully constructed plasmids were transformed into Escherichia coli BL21 competent cells for induced expression and affinity purification of the target protein, and the expression of the target protein was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Thirty-six female BALB/c mice were randomly divided into an immunization group and a control group, of 18 mice in each group. Mice in the immunization group were injected with 4 × protein and Freund’s adjuvant, and animals in the control group were administered with phosphate-buffered saline and Freund’s adjuvant. Immunizations were administered three times at two-week intervals. Blood samples were collected from the orbital sinus 0, 2, 4, 6 weeks following immunization, and serum samples were isolated. The levels of serum IgG antibodies were measured using ELISA. In the final detection, the levels of IgG antibody subtypes were also assessed. Two weeks after the final immunization, ten mice from each group were sacrificed, and spleens were aseptically harvested to prepare splenocyte suspensions. Splenocyte suspensions from five mice were used to measure cell proliferation using the CCK8 assay, while those from the other five mice were used for analysis of cytokine levels using flow cytometry. Mouse peripheral blood was sampled two weeks following final immunization, and proportions of mouse T cell subsets were detected using flow cytometry in five mice from each group. Eight mice were sampled from each group two weeks following final immunization and challenged intraperitoneally with 100 tachyzoites of the T. gondii RH strain. Then, the burdens of T. gondii in organs of 3 mice were examined using real-time quantitative fluorescence PCR (qPCR) assay, and the survival of other 5 mice were monitored daily and survival curves were plotted, statistical analysis was performed using GraphPad Prism 8 software. Results The PCR amplification product of the 4 × gene was 3 576 bp, which was consistent with the expected size. Recombinant plasmids showed expected band sizes upon characterization with PCR assay and enzymatic digestion. The target protein was successfully expressed and purified, with prominent bands observed at M_r 110 000 by both SDS-PAGE and Western blotting. ELISA results showed that the IgG antibody levels (A₄₅₀ values) in the immunized group at weeks 0, 2, 4, and 6 post-immunization were 0.079 ± 0.004, 0.759 ± 0.179, 1.670 ± 0.243, and 2.461 ± 0.056, respectively. The corresponding values for the control group were 0.080 ± 0.006, 0.067 ± 0.009, 0.080 ± 0.014, and 0.076 ± 0.011, respectively. Except for week 0 (t = 0.05, P > 0.05), the IgG levels in the immunized group were significantly higher than those in the control group (t = 9.88, 23.33, 34.66; all P < 0.05). At week 6 post-immunization, the absorbance values of IgG1 and IgG2a antibodies were 3.202 ± 0.401 and 3.725 ± 0.066 in the immunization group and 0.082 ± 0.003 and 0.059 ± 0.017 in the control group (t = 13.73, 96.83; both P < 0.05), respectively. Flow cytometry detected that the levels of IFN-γ, IL-2, IL-12, IL-4, and IL-10 were (4 998.52 ± 2 131.24), (5.76 ± 1.02), (1.38 ± 0.86), (28.83 ± 1.64), and (3 376.57 ± 218.48) pg/ml in the immunization group and (8.90 ± 0.17), (3.05 ± 0.50), (0.06 ± 0.13), (11.18 ± 1.58), (13.87 ± 3.55) pg/ml in the control group (t = 4.89, 4.61, 3.06, 15.93, 30.30; all P < 0.05), respectively, and the proliferation level of splenocytes was 3.286 ± 0.552 in the immunization group and 1.251 ± 0.157 in the control group (t = 9.15, P < 0.05). The proportions of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were (31.15 ± 2.83)% and (18.06 ± 2.82)% in the immunization group and (26.82 ± 1.17)% and (9.33 ± 1.22)% in the control group (t = 3.16, 6.36; both P < 0.05), respectively. RT-qPCR assay detected that parasite burdens were (206.7 ± 16.0), (100.2 ± 15.4), and (7.1 ± 2.0) fg/mg in the mouse liver, spleen, and lungs in the immunization mice, and (20 086.0 ± 1 310.0), (26 254.0 ± 9 658.0) and (5 300.0 ± 741.2) fg/mg in the control group (t = 26.29, 4.69, 12.37; all P < 0.05). All mice in the control group died within 8 days following challenge, in the immunized group, only one mouse survived throughout the 30 d observation period, with a significantly prolonged survival time (χ² = 7.47, P < 0.05). Conclusion The four-antigen fusion protein vaccine may induce effective humoral and cellular immune responses in mice and present a significant protective effect against acute T. gondii infection.

REVIEWS

Advances in the mechanism underlying the interaction of Toxoplasma gondii and nematodes co-infections

LV Fangli, LIN Xincheng

2025, 43(4):  562-566.  doi:10.12140/j.issn.1000-7423.2025.04.017

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Parasitic co-infection is common in the natural world, which may affect both host immune responses and the pathogenesis of parasites. Protozoa and helminths are found to induce different immune responses, and protozoa and helminths co-infections may affect host immune response to these parasites and the outcomes of infections. Toxoplasma gondii is a neurotropic protozoan parasite causing persistent subclinical neuroinflammations, which is associated with psychiatric and neurodegenerative diseases, as well as other diseases. However, there has been limited knowledge on the mechanisms underlying the interaction of T. gondii and nematodes co-infections until now. This review provides an overview of the interactions and regulatory mechanisms of T. gondii co-infections with Toxocara spp., Trichinella spiralis, Heligmosomoides spp. and Nippostrongylus brasiliensis in human or animal hosts.

Research progress on the pathogenesis and control of clonorchiasis

LI Pange, FU Jinghua, XU Minjun

2025, 43(4):  567-574.  doi:10.12140/j.issn.1000-7423.2025.04.018

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Clonorchis sinensis is an important zoonotic parasite with fish and shrimp as intermediate hosts and mammals as definitive hosts, which mainly causes digestive diseases, such as cholangiocarcinoma. With improvements of residents’ consumption level and changes of consumption structure in China, the number of human clonorchiasis cases caused by consuming raw or half cooked fish reaches up to more than 13 million in China, and clonorchiasis has become an urgent problem to be solved in the field of food safety and public health. This article reviews the latest international and national advances in the pathogenesis and prevention and control measures of C. sinensis infection in recent years, so as to provide insights into surveillance, prevention and control of this pathogen.

Currrent status and control strategy of Capillaria hepatica infection

LIU Pingguo, YIN Jiaxiang

2025, 43(4):  575-580.  doi:10.12140/j.issn.1000-7423.2025.04.019

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Hepatic capillariasis is a zoonotic parasitic disease caused by Capillaria hepatica infection in the liver of mammals, with rodents serving as important transmission and reservoir hosts. With the recent tourism development and increased human activities, there is a significant increase in the risk of human C. hepatica infection via rodents due to an increased opportunity of human contacts with rodents. This article reviews the pathogenesis, current prevalence of human infection, diagnosis and treatment of C. hepatica, and prevalence of infection in rodents, and proposes the control strategy for hepatic capillariasis based on the One Health concept, so as to achieve the shift of hepatic capillariasis management to earlier stages.

Heterogeneity of the immune microenvironment in hepatic alveolar echinococcosis and its impact on disease progression

SHI Dalin, PANG Mingquan, WU Defang, XU Xiaolei, WANG Cheng, FAN Haining

2025, 43(4):  581-587.  doi:10.12140/j.issn.1000-7423.2025.04.020

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Hepatic alveolar echinococcosis (HAE), a severe parasitic disease caused by Echinococcus multilocularis larvae infections in the liver, is characterized by high rates of disability and mortality and poses a great threat to both human and animal health. It has been recently found that the immune microenvironment in HAE exhibits considerable heterogeneity, which not only influences host immune responses to parasites, but also poses important impacts on disease progression and clinical manifestations. This article reviews the latest advances in the immune microenvironment of HAE from the perspective of immune microenvironment heterogeneity, discusses the impact on disease progression and proposes future research directions.

SHORT COMMUNICATIONS

Prevalence and spatial distribution of Fasciola spp. in cattle in Fujian Province

JIANG Dianwei, CAI Wuwei, ZHENG Dan, XIE Hanguo, GAO Lanlin

2025, 43(4):  588-591.  doi:10.12140/j.issn.1000-7423.2025.04.021

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To investigate the prevalence and geographical distribution of Fasciola spp. in bovine in Fujian Province, 2 to 3 counties were selected from 9 cities and one comprehensive experimental area in Fujian Province from May 2023 to May 2024 as bovine stool sampling sites. Allocate sampling quantities based on the proportion of bovine in each county to the total number of bovine in the province, with one fecal sample taken from each bovine. The Fasciola eggs in bovine stool samples were collected using the precipitation method for morphological characterization. Ten Fasciola eggs were randomly sampled from positive fecal samples in each city (district), and genomic DNA was extracted from eggs for PCR amplification of ribosomal internal transcribed spacer region 2 (ITS2) gene. All statistical analyses were performed using the software SPSS 20.0. A total of 502 bovine stool samples were collected, and there 110 samples detected with Fasciola eggs, with 21.9% (110/502) prevalence of infections. The detected Fasciola eggs were elongated in shape, light yellow brown in color, and contained a small cap at one terminal. The Fasciola infection rates of the 10 cities (districts) from high to low are Pingtan District (52.8%, 19/36), Fuzhou City (42.5%, 34/80), Longyan City (37.9%, 25/66), Ningde City (23.5%, 8/34), Putian City (16.0%, 8/50), Zhangzhou City (12.8%, 6/47), Quanzhou City (10.3%, 6/58), Xiamen City (9.3%, 3/32), Sanming City (1.7%, 1/59), Nanping City (0/40), and there was a significant difference in the prevalence of Fasciola infections among the 10 cities (districts) (Fisher’s exact test, P < 0.01). Specific bands were amplified at 550 bp in Fasciola-infected bovine stool samples from 9 cities (districts). Spatial analysis showed that geographical distribution of Fasciola spp. was scattered in bovine in Fujian Province, with a high coverage rate and a landscape of “high in the northwestern region and low in the southeastern region”, with hotspots identified in Pingtan District, Fuzhou City, and Longyan City (prevalence of infection > 36.0% to 54.0%). The findings showed that the geographical distribution of Fasciola spp. is widespread in bovines in Fujian Province, with relatively high prevalence in livestock. There is a risk of infection with bovine fasciolosis among individuals living in Fujian Province.

Morphological and molecular identification of Cathaemasia hians in Ciconia nigra in Inner Mongolia

LI Jian, LI Fenqi, GAO Nan, LIU Qiang, ZHOU Qing’an, LI Chunfu, HU Wei

2025, 43(4):  592-595.  doi:10.12140/j.issn.1000-7423.2025.04.022

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Trematodes were collected from the pharynxes and oesophagi of Ciconia nigra at the Daqingshan Wildlife Park in Hohhot, Inner Mongolia on August 2024. Ten morphologically intact adult worms were sampled and stained with hydrochloric carmine for morphological observation. Genomic DNA was extracted from three adult worms, and the internal transcribed spacer 2 (ITS2) gene was amplified using PCR assay and sequenced. ITS2 gene sequences were aligned using BLAST in GenBank and a phylogenetic tree was built using the software MEGA 11.0 with the neighbor-joining method. A total of 157 trematodes were collected from pharynxes of 10 C. nigra, with mean infection intensity of approximately 16 (range, 8 to 33) worms per bird. The trematodes appeared a leaf-like shape, with dendroidal testes and anterior ovary, which matched the morphological characteristics of Cathaemasia hians. PCR assay and paired-end sequencing yielded 3 ITS2 gene sequences, with 425 bp in length, which was identical to the ITS2 sequence of C. hians. The ITS2 sequence had been deposited in GenBank under accession number PQ899708. Sequence alignment showed that the ITS2 gene sequence had a single C→T substitution at position 365 with the Czech isolate of C. hians, and phylogenetic analysis revealed that the ITS2 gene sequence was clustered with the reference sequence of C. hians in GenBank into the same clade (98% bootstrap value), indicating that the ITS2 gene sequence had an intra-species monophyly and a relatively stable genetic background.

Detection of Schistosoma japonicum cercariae using environmental DNA assay

WEN Yusong, XU Hui, QIU Tingting, ZHAO Qin, LI Jie, XU Jianrui, ZENG Xiaojun, Ding Sheng, LI Zhaojun

2025, 43(4):  596-599.  doi:10.12140/j.issn.1000-7423.2025.04.023

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To investigate the feasibility and sensitivity of environmental DNA (eDNA) assay for detection of Schistosoma japonicum cercariae, cercariae shed from S. japonicum-infected Oncomelania hupensis snail were collected,with 1, 5, 10, 15, 20, and > 30 cercariae as experimental groups and 0 cercaria as the control group. After standing for 24 hours, cercariae were filtered and water samples were collected. Then, eDNA was enriched using membrane filter with 0.22 μm in pore size, and extracted using the Water DNA Isolation Kit. The Real-time fluorescence quantitative PCR (qPCR) assay was performed with S. japonicum mitochondrial cytochrome c coxidase subunit 1 (cox1) and 16S rRNA genes as specific primers to detect eDNA in each group. The cycle threshold (Ct value) was recorded, and the associations of Ct value with cercariae density and eDNA concentration were examined using Spearman correlation analysis. qPCR assay detected S. japonicum cercariae DNA in all experimental groups of different densities of cercariae with the primers cox1 and 16S rRNA. The DNA concentrations were 2.4, 1.0, 0.4, 1.5, 1.3, and 9.4 μg/ml, respectively. Spearman correlation analysis revealed that the cercariae density significantly negatively correlated with the Ct value with the primer cox1 gene (r = -0.886, P < 0.05), and negatively correlated with the Ct value with the primer 16S rRNA gene (r = -0.771, P > 0.05). The eDNA concentration negatively correlated with the Ct value with the primer cox1 gene (r = -0.314, P > 0.05) and the primer 16S rRNA gene (r = -0.200, P > 0.05). These findings suggest that eDNA assay is feasible for detection of S. japonicum cercariae with high timeliness, which has the value for early warning of environments at a high risk of S. japonicum infections.

CASE REPORT

An imported case of pulmonary paragonimiasis heterotremus

ZHANG Mingfang, JI Yuanhao, GU Shengli, KUAI Huifen, QUAN Bin, HOU Weishun, YANG Jinsun

2025, 43(4):  600-602.  doi:10.12140/j.issn.1000-7423.2025.04.024

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A 27-year-old female patient from Laos, with a history of consuming raw freshwater crabs, had been residing in Wuhu City, Anhui Province for the past six months. On April 22, 2025, she presented to the First Affiliated Hospital of Wannan Medical College with a one-year history of recurrent cough and expectoration. Her chest CT revealed infectious lesions in both lungs with multiple cavities, cystic changes, and a small amount of pleural effusion on both sides. The blood routine test showed elevated absolute eosinophil count (1.2 × 10⁹/L) and percentage (12.9%). Metagenomic next-generation sequencing (mNGS) performed on the patient’s bronchoalveolar lavage fluid detected 446 sequences of Paragonimus heterotremus. Eggs were identified upon microscopic examination of the patient’s sputum, leading to a definitive diagnosis of pulmonary P. heterotremus infection. The patient was treated with praziquantel (orally, 1 g per dose, three times daily for three consecutive days) as an antiparasitic therapy, along with cough-relieving and expectorant care. Her cough symptoms significantly improved compared to before, and follow-up examination revealed a reduction in the eosinophil percentage to 3.4%. Reports of P. heterotremus infection are rare in China. This case suggests that medical personnel should consider the possibility of parasitic diseases such as paragonimiasis when encountering patients with chronic cough that does not respond to conventional treatment.