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    30 August 2024, Volume 42 Issue 4
    EXPERT VIEWPOINT
    Interpretations of some terminology of Trichinella and trichinellosis
    CUI Jing, WANG Zhongquan
    2024, 42(4):  427-432.  doi:10.12140/j.issn.1000-7423.2024.04.001
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    Trichinellosis is a serious zoonotic parasitosis. During the teaching and studies, some Trichinella and trichinellosis terminology (including Latin names and Chinese names) can be confusing. Complete interpretations of these terms have not been found in the relevant textbooks and monographs. In this article, some Trichinella and trichinellosis terminologies were interpreted to provide references for teaching and studies of Trichinella and trichinellosisin China.

    ORIGINAL ARTICLES
    Identification of caspase 8 of Haemaphysalis longicornis and its role in resisting Babesia microti infection
    ZHU Haotian, ZHOU Yongzhi, CAO Jie, WANG Ya’nan, ZHANG Houshuang, XU Qianming, ZHOU Jinlin
    2024, 42(4):  433-438.  doi:10.12140/j.issn.1000-7423.2024.04.002
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    Objective To identify the Haemaphysalis longicornis caspase 8 (cas8) gene, and to investigate its role in resisting Babesia microti infection, to lay the foundation for development of vaccine interrupting transmission of B. microti. Methods The RNA of the H. longicornis was extracted and reverse transcribed into cDNA, the cas8 was amplified by PCR and sequenced, and sequence alignment was performed in GenBank with BLAST. Using MEGA software, a phylogenetic tree based on the cas gene was constructed with the neighbor-joining method. The H. longicornis nymph that had bitten B. microti infected-mice were assigned as infection group, while the same batch of ticks that had bitten normal mice as the control group. Nine ticks from each group were collected for extraction of RNA to reverse transcribe into cDNA. Quantitative PCR (qPCR) was used to analyze the relative transcriptional differences of cas8 between the infection group and the control group ticks. The total protein of H. longicornis nymph from the infection group and the control group were extracted for analysis of relative expression level of by Western blotting. Double stranded RNA (dsRNA) of cas8 gene and luciferase gene were synthesized and microinjected into the tick nymphs for RNA interference (RNAi) (RNAi group and control group, respectively). Upon 12-24 hours post microinjection, the ticks with good activity were picked and fed with B. microti infected-mice by biting. Immediately after fully fed of blood, the ticks were collected to detect relative transcriptional level of cas8 gene with qPCR, and to detect the 18S rRNA of B. microti in H. longicornis by probing with real-time PCR. The comparison of relative transcription levels and protein expression levels of cas8 gene between the infection group and the control group, and content of B. microti 18S rRNA in the RNAi group and control group were performed using t-test. Results The cas8 gene of H. longicornis is 1 377 bp in length, and the accession number obtained by submitting the sequence to GenBank is PP407944. The predicted amino acid sequence is 21.26% and 61.24% in consistence with the amino acid sequences of H. longicornis CAS8 (GenBank: ABG48761) and Rhipicephalus haemaphysaloides CAS8 (GenBank: ALQ43547.1) from GenBank, respectively. The phylogenetic tree analysis showed that the cas8 were not at the same branch with the H. longicornis cas sequences (ABG48665.1, ABG48761.1) from GenBank, indicating a distant genetic relationship; while the cas8 was at the same branch with the cas8 of R. haemaphysaloides (GenBank: ALQ43547.1), indicating a relatively closer genetic relationship. The qPCR analysis results showed that the relative transcription level of the cas8 in infection group was 0.562 ± 0.036, which was higher than that in the control group (0.198 ± 0.071) (t = 7.910, P < 0.01). The Western blotting results showed that the relative expression level of CAS8 protein in the H. longicornis in infection group was 0.460 ± 0.013, which was higher than that in the control group (0.346 ± 0.007) (t = 9.368, P < 0.01). After silencing cas8 by RNAi, the relative transcription level of the cas8 in RNAi group was 0.036 ± 0.003, which was lower than that in the control group (0.081 ± 0.006) (t = 10.680, P < 0.01). The content of B. microti 18S rRNA in the ticks in RNAi group was 3.35 × 106 copies/μl, which was higher than that in the control group (1.35 × 106 copies/μl) (t = 4.570, P < 0.05). Conclusion CAS8 is an apoptosis related molecule, which can resist the infection of B. microti to H. longicornis. It may be used as a candidate target molecule for the transmission blocking vaccine of B. microti.

    Metagenomic analysis and potential assessment of Hyalomma asiaticum in the distribution area of Przewalski’s horses
    ZHANG Yu, ZHANG Ke, LIU Jiawei, WANG Anqi, TUAN Yong, ZHANG Dong, YAN Liping, LI Kai
    2024, 42(4):  439-446.  doi:10.12140/j.issn.1000-7423.2024.04.003
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    Objective To evaluate the potential risk of disease transmission by Hyalomma asiaticum in the distribution area of Przewalski’s horses, investigate the metagenomic characteristics and conduct pathogen analysis of male and female ticks. Methods In April 2022, tick samples were collected using the “waiting for ticks” method in the Kalamaili Mountain Ungulate Nature Reserve in Xinjiang. The ticks were morphologically identified under a stereomicroscope, and DNA of 48 ticks (24 males and 24 females) was extracted for molecular identification by PCR amplification of the mitochondrial cytochrome C oxidase subunit 1 (COⅠ) sequence. Metagenomic sequencing of H. asiaicum was conducted by grouping the ticks according to sex. The non-redundant sequences were compared to the non-redundant protein (NR) database to analyze the composition of the microbial communities carried by the ticks. Additionally, comparisons were made with the Kyoto encyclopedia of genes and genomes (KEGG) and the antibiotic resistance genes database (ARDB) to obtain the annotations for gene function and the antibiotic resistance functions in ticks and tick-borne pathogens. The data were analyzed using t-test. Results A total of 124 ticks were collected and morphological identification revealed that 119 were H. asiaicum adult. The PCR amplification result showed that the positive production with a length of 700 bp were amplified from tick DNA and the sequences were 99%-100% identical to H. asiaicum (Genbank: MH459386.1). After quality control filtering, a total of 469 327 812 sequence reads were obtained from metagenomic sequencing, and open reading frame prediction yielded 836 843 to 1 094 994 sequences. The NR database comparison revealed that the bacterial community abundance carried by H. asiaicum accounted for 99.13% of the total community abundance, with bacteria from 32 phyla and 2 040 species identified. The predominant phyla are Proteobacteria and Actinobacteria, accounting for 51.52% and 44.35% of the bacterial community abundance, respectively. The dominant species is Anaplasma phagocytophilum, accounting for 16.35% of the bacterial community abundance. The viral community abundance accounts for 0.004% of the total community abundance, with viruses from 5 phyla and 21 species identified. There were no significant differences in the richness and diversity of bacterial and viral communities between female and male ticks (t = -1.180、-1.729, both P > 0.05). KEGG gene function analysis revealed that the highest proportion of genes in H. asiaticum were involved in metabolism (54.38%), with the primary functional categories include amino acid metabolism, carbohydrate metabolism, and membrane transport. A total of 11 352 pathways were identified, 154 of which exhibited statistically significant differences between female and male ticks (t = -2.348, P < 0.05). ARDB analysis revealed that H. asiaticum carried 154 antibiotic resistance genes, comprising 49 different types. The major type of resistance gene was Baca (62.53%), and the main class of antibiotics was glycopeptides. The majority of resistance genes were associated with multi-drug resistance, including Mexf, Mexb, Emrd, Mexw and others. The resistance of Emrd gene in female ticks were higher than male (t = -7.558, P < 0.05). Conclusion This study revealed the major bacterial and viral communities carried by the H. asiaticum at the metagenomic level, along with multidrug resistance-related antibiotic resistance genes. Female ticks and their associated pathogens exhibited higher species richness and gene abundance.

    Echinococcus multilocularis metacestode regulates its own growth through hepatic p38MAPK signaling pathway
    XU Gang, MAO Yi, LI Jiang, ZHANG Hongwei, ZHANG Yongguo, WU Xiangwei, PENG Xinyu, SUN Hong, YANG Jing, CHEN Qian, ZHANG Shijie
    2024, 42(4):  447-453.  doi:10.12140/j.issn.1000-7423.2024.04.004
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    Objective To investigate the influence of Echinococcus multilocularis on the p38MAPK signaling pathway in liver tissue and the affect of p38MAPK expression level on E. multilocularis metacestodes. Methods E. multilocularis protoscolices were collected from the abdominal cavity of preserved gerbils. C57BL/6J mice aged 4-6 weeks were randomly assigned into three groups as infected group, uninfected group, and inhibitory group, with 10 mice each group. The inhibitory group mice were injected via liver portal vein with SB202190 solution (5 mg/kg, in mixed volvent of 20% DMSO and 80% sterile saline), while the uninfected and infected groups received the solvent only, once a week, for total 4 weeks. Subsequently, both the infected and inhibitory groups were given by liver subcapsular injection with 500 protoscolices and raised for 8 weeks. After euthanasia, the mice livers were collected and weighed to calculate weight ratio (liver mass/ body mass). Liver tissues adjacent to (within 0.3 cm) and distant from (1 cm away) the metacestode vesicles were collected. These liver samples were embedded with paraffin and sectioned, and then stained with hematoxylin-eosin (HE), periodic acid-schiff (PAS), and Masson’s trichrome for analyzing the range of inflammatory bands and hepatic fibrosis. Utilizing the TRIzol method, RNA was extracted from the liver tissues to detect the expression levels of MAPK14 gene by quantitative PCR (qPCR). Subsequently, liver tissues adjacent to the vesicles from each group were examined for the expression levels of p38MAPK and p-p38MAPK using Western blotting and immunohistochemical methods, respectively. Results The livermass/body mass weight ratio in the uninfected group was (6.49 ± 0.19)%, while it was increased to (6.80 ± 0.33)% in the infected group (t = 2.74, P < 0.05). The liver size in the uninfected group was (5.27 ± 0.34) cm3, whereas it increased to (5.80 ± 0.49) cm3 in the infected group (t = 2.83, P < 0.05). HE staining indicated the absence of inflammatory bands in the uninfected group, with the infected and inhibitory groups showing inflammatory band areas of (51.2 ± 14.0)% and (23.8 ± 9.8)%, respectively. The inhibitory group displayed a significant decrease in hepatic necrotic foci and inflammatory bands compared to the infected group (t = 3.92, P < 0.01). PAS staining revealed that the area of purple-red stained regions accounted for (1.3 ± 0.3)% in the uninfected group, (7.4 ± 1.8)% in the infected group, and (4.5 ± 0.4)% in the inhibitory group. The inhibitory group showed a significant reduction in the area of purple-red staining compared to the infected group (t = 3.82, P < 0.05). Masson’s trichrome staining demonstrated an absence of blue-stained areas in the uninfected group, indicative of the absence of collagen deposition. In contrast, the infected and inhibitory groups exhibited blue-stained areas representing (34.9 ± 4.1)% and (16.3 ± 2.8)% of the total tissue area, respectively. The extent of the blue-stained areas was significantly reduced in the inhibitory group compared to the infected group (t = 9.16, P < 0.01). qPCR analysis indicated that the relative expression of MAPK14 in liver tissues adjacent to E. multilocularis lesions in the infected group was significantly higher, with a level of 7.14 ± 2.23 (t = 6.13, P < 0.01), compared to tissues distant from the lesions (1). The infected group also demonstrated an elevated MAPK14 expression level of 3.17 ± 0.68 (t = 7.14, P < 0.01) relative to the uninfected group (1). The inhibitory group displayed a substantial decrease in MAPK14 expression levels adjacent to the lesions, with a relative expression of 0.07 ± 0.01, significantly lower than that of the infected group (1) (t = 126.83, P < 0.01). Western blotting data indicated that the ratio of p38MAPK to its phosphorylated form, p-p38MAPK, was significantly different among the groups. In the uninfected group, the relative expression levels of p38MAPK and p-p38MAPK, as well as p38MAPK/p-p38MAPK values were 0.80 ± 0.08, 0.67 ± 0.11, and 0.74 ± 0.09, respectively. For the infected group, the ratio increased to 0.97 ± 0.14, 0.87 ± 0.09, and 0.91 ± 0.14, respectively. The inhibitory group exhibited a markedly reduced ratio to 0.41 ± 0.07, 0.20 ± 0.07, and 0.49 ± 0.21, respectively, when compared to the infected group (t = 7.97, 13.32, 3.74, all P < 0.01). Immunohistochemical results showed p38MAPK scores of 4 (4, 4) for the uninfected group, 6 (6, 9) for the infected group, and 4 (2, 4) for the inhibitory group. The p-p38MAPK scores were 4 (4, 6), 9 (8, 9.75), and 6 (6, 6), respectively. Compared to the infected group, the inhibitory group exhibited a significant decrease in the molecular expression levels of both p38MAPK and p-p38MAPK in liver tissues adjacent to the lesions (Z = -3.00, -3.11, both P < 0.01). Conclusion E. multilocularis metacestode could promote the expression of p38MAPK and its phosphorylation in the liver tissue adjacent to the vesicles, while the p38MAPK signaling pathway in adjacent liver tissue may involve in maintaining the growth and invasive ability of metacestode.

    Application of convolutional neural networks in ultrasound classification of hepatic cystic echinococcosis
    REN Yan, SONG Tao, SHANG Feng, WU Miao, WANG Zhengye, WANG Xiaorong
    2024, 42(4):  454-460.  doi:10.12140/j.issn.1000-7423.2024.04.005
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    Objective To evaluate the application value of convolutional neural network (VGG19) model in the ultrasound diagnosis of hepatic cystic echinococcosis (HCE). Methods The ultrasound images of patients with hepatic cystic echinococcosis (HCE, including CE1-CE5 types) and patients with non-echinococcosis focal liver lesions (NHFLL) in the First Affiliated Hospital of Xinjiang Medical University from January 2012 to December 2022 were retrospectively collected. The VGG19 model was used to determine the image diagnosis of 6 types of hepatic focal space occupying lesions (CE1-CE5, NHFLL), and the percentage of each type determined was compared. When VGG19 misclassified HCE and NHFLL, the general demographic information and relevant clinical data of patients were compared. The ultrasound images were randomly divided into two groups based on the principle of essentially consistent proportion of different types, of which manual classification summary were performed by 2 junior ultrasound physicians and 2 senior ultrasound physicians on one group each selected randomly. The diagnostic accuracy of the model made by junior and senior ultrasound physicians were compared. The diagnostic performance of VGG19 was evaluated using confusion matrix, precision, recall, specificity, and F1 score. Descriptive analysis was conducted using contingency tables to describe the count data, and chi-square test, Fisher’s exact probability method and paired card square test were used for comparative analysis of differences. Results Among the 871 HCE cases, there were 203 cases of CE1, 227 of CE2, 110 of CE3, 159 of CE4, and 172 of CE5. The 600 NHFLL cases include, 300 cases of hepatic cysts, 150 of hepatic calcified lesions, and 150 solid hepatic occupying lesions (100 cases of hepatic hemangioma, 25 of hepatoma, and 25 of liver abscess). The overall accuracy of the VGG19 model was 82.0%, the recall rate was 87.9%, and the F1 score was 84.3%. The overall accuracy rate of VGG19 model was 86.2% (1 268/1 471), and the accuracy rates of each type from high to low were CE5 (95.3%, 164/172), CE4 (91.2%, 145/159), CE3 (89.1%, 98/110), CE1 (84.7%, 135/159), CE2 (84.6%, 192/227) and NHFLL (82.8%, 497/600), respectively. A total of 203 cases were misdiagnosed, and the misdiagnosis rate was 13.8% (203/1 471). Among them, 100 cases were misdiagnosed between HCE types, including 31 cases of CE1, 35 cases of CE2, 12 cases of CE3, 15 cases of CE4, and 8 cases of CE5. 103 cases of NHFLL were misdiagnosed as HCE, including 68 cases of hepatic cyst, 17 cases of liver calcification, 17 cases of liver hemangioma, and 1 case of liver abscess. No case of liver cancer was misdiagnosed. There were statistically significant differences in age, residential area, history of contact with dogs, education level and province between HCE patients and NHFLL patients misdiagnosed by VGG19 (χ2 = 55.116, 24.197, 35.834, 14.069, 11.918, all P < 0.05), but there was no significant difference in gender (χ2 = 0.047, P > 0.05). The overall diagnostic accuracy of VGG19 model (86.2%, 1 268/1 471) was higher than that by junior doctors (81.2%, 1 195/1 471) (P < 0.05), lower than that by senior physicians (92.3%, 1 358/1 471) (P < 0.05). Conclusion The VGG19 model can identify the 5 types of HCE and non echinococcal NHFLL, its diagnostic accuracy is lower than that by senior sonographers, but higher than that by junior sonographers. The model is expected to be promoted to primary hospitals to assist correction of ultrasound diagnosis in combination with clinical information.

    Analysis on influencing factors of hospitalization costs of echinococcosis surgery patients in designated echinococcosis hospital in Gansu Province
    XU Dan, YAN Jun, YAN Huan, HE Hongfeng, LIANG Tianhu, ZHAO Junxian, CHEN Yaolong, LI Xun
    2024, 42(4):  461-468.  doi:10.12140/j.issn.1000-7423.2024.04.006
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    Objective To analyze the hospitalization cost of echinococcosis surgery patients and its influencing factors in Gansu provincial designated echinococcosis hospital, and to provide basis for effieicent management of the medical cost for echinococcosis. Methods Data of echinococcosis patients who underwent surgery at provincial designated hospital in Gansu Province from January 2014 to December 2023 were collected. The data included patients’ basic information (hospitalization year, gender, occupation, age, medical insurance type, ethnicity), clinical information (admission pathway, whether transferred from another department, length of hospital stay, type of echinococcosis, disease outcome, presence of complications, location of cyst, type of surgery), and hospitalization cost data (total hospitalization cost, surgery cost, western medicine cost, consumables cost, diagnostic cost, other costs). Data analysis was conducted using the SPSS 29.0 statistical software. Median was used to represent the hospitalization cost. In single factor analysis, Mann-Whitney U test was used for comparison between two groups, and Kruskal-Wallis H test for comparison among multiple groups for non normal distribution data. Multivariate analysis was performed using multiple linear regression. Results A total of 540 echinococcosis surgery patients’ data were collected, including 504 patients with cystic echinococcosis and 36 patients with alveolar echinococcosis; 280 females and 260 males, with a total combined hospitalization cost of 20 809 800 yuan, a per capita hospitalization cost of 38 528.41 yuan, and median hospitalization cost of 33 732.87 yuan. In 2014 versus 2023, the hospitalization costs for patients with echinococcosis per capita increased from 31 276.59 yuan to 45 902.34 yuan. The median hospitalization costs for male and female cases were 35 232.35 and 31 891.16 yuan, respectively, and the difference was statistically significant (Z = -2.343, P < 0.05); the median hospitalization cost for patients ≥ 61 years old was the highest, 35 233.75 yuan, and the cost difference between different ages was statistically significant (P < 0.05); the median hospitalization cost for individual patients was the highest, 39 843.04 yuan, and the difference in hospitalization costs between different occupations was statistically significant (P < 0.05); the median hospitalization cost for Dongxiang patients was the highest at 65 401.29 yuan, and the difference in hospitalization costs between different ethnic groups was statistically significant (P < 0.05); the median hospitalization cost for patients transferred to ICU was 62 720.04 yuan, higher than the 32 526.89 yuan for patients who were not transferred to the department, and the difference was statistically significant (Z = -7.440, P < 0.05); the median hospitalization cost of patients with hospitalization time ≥ 21 d was the highest at 54 433.71 yuan, and the difference in hospitalization cost between different hospitalization times was statistically significant (P < 0.05); the median hospitalization cost of patients with multilocular echinococcosis was 43 949.85 yuan, higher than that of patients with fine-grained echinococcosis at 33 555.82 yuan (Z = -3.317, P < 0.05); the median hospitalization cost of transferred patients was the highest at 104 536.50 yuan, and the difference in hospitalization costs for different types of disease transfer was statistically significant (P < 0.05); the median hospitalization cost among patients with complications was 46 855.89 yuan, higher than that of 32 476.43 yuan for patients without complications (Z = -6.060, P < 0.05); the median hospitalization cost of patients with hepatic echinococcosis was 32 433.02 yuan, which was lower than that of patients with multi-organ echinococcosis, which was 43 550.83 yuan (Z = -4.933, P < 0.05); the median hospitalization cost of patients who underwent liver transplantation was the highest, which was 170 030.84 yuan, and the difference in hospitalization costs between different surgical procedures was statistically significant (P < 0.05). The results of multivariate linear regression analysis showed that the main factors influencing the cost of hospitalization for patients undergoing surgery for echinococcosis were year of hospitalization (t = 5.937, P < 0.01), age (t = 3.349, P < 0.01), occupation (t = 2.238, P < 0.05), whether or not to transfer to a different department (t = 8.778, P < 0.01), length of hospitalization (t = 7.346, P < 0.01), type of echinococcosis (t = 6.049, P < 0.01), the presence or absence of complications (t = 5.967, P < 0.01), location of cyst (t = 3.767, P < 0.01). Conclusion The per capita hospitalization cost of echinococcosis surgery patients in provincial designated hospital increased year by year from 2014 to 2023, The main factors influencing of hospitalization costs include year, age, occupation, whether or not to transfer to a different department, length of hospitalization, type of echinococcosis, the presence or absence of complications and location of cyst.

    Analysis on surveillance results of clonorchiasis in Zhejiang Province from 2013 to 2022
    CHAI Yingzhi, CHEN Hualiang, YU Kegen, ZHANG Xuan, WANG Xiaoxiao, ZHANG Jiaqi, XU Wenjie, RUAN Wei
    2024, 42(4):  469-474.  doi:10.12140/j.issn.1000-7423.2024.04.007
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    Objective To ascertain the endemic status of Clonorchis sinensis in Zhejiang Province. Methods From 2013 to 2017, one county (city, district) which had clonorchiasis cases reported before in Zhejiang Province was selected as the regular surveillance site and 3 townships in each site were selected for monitoring each year. No less than 100 permanent residents over age 3 in each township were selected as monitored objects. Venous blood samples were collected, and serum C. sinensis antibody was detected by enzyme-linked immunosorbent assay (ELISA). The positive rate of antibodies in the monitored objects was calculated. And questionnaire surveys were conducted on the factors affecting the infection of C. sinensis from 2013 to 2015. From 2018 to 2022, Ningbo City was selected as the fixed surveillance site and 7-9 counties (cities, districts) were selected as the mobile surveillance sites. Each surveillance site was divided into 5 areas as geographically at east, west, south, north and centre. In each area, one administrative village was randomly selected from one township for cluster sampling of no less than 200 permanent residents over age 3. Fecal samples (over 30 g) were collected for examining C. sinensis eggs using the modified Kato-Katz thick smear method. The infection rate of the monitored object was calculated. Freshwater fish from natural water were collected in regular surveillance sites from 2013 to 2017 and in the fixed surveillance site and a mobile surveillance site from 2018 to 2022. The infection of encysted metacercaria was detected by pressing method after identified the species. From 2018 to 2022, the fecal samples of cats, dogs and pigs were collected in surveillance sites which monitoring the intermediate hosts, and examined C. sinensis eggs using the modified Kato-Katz thick smear method. SPSS 19.0 was used for data statistical analysis. Infection rates were compared between groups using the chi-squared test. Results From 2013 to 2017, a total of 1 516 people were examined in Zhejiang Province and antibody positive rate was 2.51% (38/1 516). A total of 52 626 people were examined from 2018 to 2022. No C. sinensis eggs were found in fecal samples and the infection rate was 0 (0/52 626). The highest antibody positive rate of 6.00% (18/300) was seen in Panan County. The antibody positive rate in different surveillance sites was statistically significant (χ2 = 21.212, P < 0.01). The highest antibody positive rate of 8.82% (6/68) was seen in the age 3-17 group. The antibody positive rate in different age groups was statistically significant (χ2 = 13.105, P < 0.05). The infection rate of C. sinensis in freshwater fish from 2013 to 2022 were 12.56% (476/3 791). The highest infection rate of 33.47% (84/251) was seen in Ninghai County in 2018. The infection rate in different surveillance sites was statistically significant (χ2 = 242.727, P < 0.01). The highest infection rate of 75.00% (21/28) was seen in Acanthobrama simoni. The infection rate in different fish species was statistically significant (χ2 = 174.750, P < 0.01). The infection rate of reservoir host was 1.17% (3/257). Conclusion The infection rate of C. sinensis in human population was considerably low in surveillance sites in Zhejiang Province, but the infected intermediate hosts and reservoir hosts still remained, which poses a potential epidemic risk and requires strengthened surveillance, prevention and health education.

    Infection and identification of Clonorchis sinensis metacercariae in Pseudorasbora parva in multiple regions of China
    WANG Jufeng, XUE Shijie, LIU Jiangyong, LAI Dehua, LUN Zhaorong
    2024, 42(4):  475-480.  doi:10.12140/j.issn.1000-7423.2024.04.008
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    Objective To understand the infection, morphological and molecular identification of Clonorchis sinensis metacercariae in Pseudorasbora parva in multiple regions of China. Methods From August 2022 to January 2024, P. parva samples were collected from 12 sampling sites of water bodies in East, South, Central, Northwest, and Southwest China. After digesting the fish, metacercariae were isolated for morphological identification. From each sampling site, individual metacercariae in infected fishes were isolated individually to extract DNA, of which 16S rDNA was amplified with PCR and sequenced. Sequence alignment was conducted using BLAST, and a phylogenetic tree was constructed using MEGA7 and Fasttree2 with the maximum likelihood method. SD rats (n = 12) and Kunming mice (n = 10) were fed with metacercariae-positive P. parva (approximately 200 metacercariae per rat and 100 metacercariae per mouse). Eggs and adult worms from fecal samples and the bile ducts were collected, respectively, for morphological and identification at 25, 90 and 300 days post-infection. Data was analyzed by Microsoft Excel 2021 software. Fisher’s exact test was used for comparison between groups. Results Out of the 665 P. parva samples examined, 277 were positive for metacercariae, resulting in an average infection rate of 41.6%. Higher infection rates of P. parva were found in Hengzhou of Guangxi (6/6), Puyang of Henan (100%, 22/22), Yantai of Shandong (98.4%, 122/124), Fuyang of Anhui (75.0%, 24/32), Hanzhong of Shaanxi (49.2%, 95/193), and Ningde of Fujian (5/19). The isolated trematode metacercariae samples were morphologically identified as of C. sinensis. PCR amplification and sequencing results showed that the amplified about 400 bp fragment matched to the sequence of 16S rDNA of C. sinensis, revealing 100% identity (GenBank accession no.: MT607652.1). Phylogenetic analysis indicated that the isolated metacercariae clustered with C. sinensis in the same branch. At 25 and 90 days post-infection, no eggs or adults were found in mouse feces or bile ducts, while eggs were found in rat feces. At 300 days post-infection, eggs were found in the feces of two rats, and adults were isolated from the bile ducts of three rats. The eggs and adults exhibited typical morphological characteristics of C. sinensis. Conclusion The present study domonstrated that C. sinensis infection at varying degrees was found in P. parva from Hengzhou, Guangxi, Puyang, Henan, Yantai, Shandong, Fuyang, Anhui, Hanzhong, Shaanxi, and Ningde, Fujian. The 16S rDNA gene combined with the morphological characteristics can effectively identify C. sinensis metacercariae in P. parva.

    Establishment of multienzyme isothermal rapid amplification assay combined with fluorescent probe for rapid detection of Schistosoma japonicum gene
    ZHANG Lesheng, WANG Qi, WANG Fengfeng, ZHU Hai, LI Qingyue, MA Xiaohe, WANG Min, WANG Yujie, WANG Tianping, CAO Zhiguo
    2024, 42(4):  481-486.  doi:10.12140/j.issn.1000-7423.2024.04.009
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    Objective To develop a method for rapid detection of specific gene fragments of Schistosoma japonicum using a multienzyme isothermal rapid amplification (MIRA) combined with fluorescent probing. Methods The S. japonicum non-long terminal repeat retrotransposons (SjR2) fragment was selected as the target sequence, and three pairs of primers and fluorescent probes were designed and synthesized to establish a fluorescent MIRA reaction system, with which, fluorescence quantitative PCR was performed, and amplification curves were plotted to compare and screen out primer pairs and probe concentrations with better amplification effects. To evaluate the sensitivity of this method, adult S. japonicum genomic DNA at different concentrations of 1 fg/μl, 5 fg/μl, 10 fg/μl, 100 fg/μl, 1 pg/μl, and 10 pg/μl, were detected, respectively. To evaluate the methd specificity, genomic DNA extracted from Paragonimus westermani, Clonorchis sinensis, C. orientalis, Gnathostoma, and Toxoplasma gondii were examined using the fluorescence MIRA method. To assess the detactable limit of serum gene DNA of the method, rabbit blood was collected from the ear vein for separating serum to prepare simulated positive serum samples containing 1 fg, 5 fg, 10 fg, 100 fg, 1 pg, and 10 pg DNA of adult S. japonicum, which were detected using the fluorescence MIRA method. Results The amplification efficiency of primer pair 1 (SjR2-1) was high, and the fluorescent product was seen at the 22nd cycle (11 min) with a maximum fluorescence value of 170 000; the probe amount at 0.6 μl/reaction displayed better fluorescence intensity with low fluorescence background. The fluorescent products were amplified at the 26th cycle (13 min) at 39 ℃. The minimum detection limit of the established fluorescence MIRA method for detecting Schistosoma adult worm DNA was 1 fg/reaction. The electrophoresis of amplification products showed that electrophoresis bands appeared when the template of Schistosoma genomic DNA was 10 pg, 1 pg, 100 fg, and 10 fg, respectively, and the detection limit was 10 fg/reaction and the product size was 186 bp. The reaction tube containing only the DNA of S. japonicum adult worm showed fluorescence amplification curves, while no significant fluorescence amplification curves were observed in the detection of genomic DNA of P. westermani, C. sinensis, C. orientalis, Gnathostomum and T. gondii. When the DNA content in the simulated positive serum of different concentrations of Schistosoma was 1 pg and 10 pg, positive amplification curves appeared, and positive fluorescence amplification signals appeared as early as the 16th cycle (within 8 min). The minimum detection limit for detecting Schistosoma adult worm DNA in simulated positive serum by this method was 1 pg/reaction. Conclusion A fluorescent MIRA method detecting specific gene fragments of S. japonicum was successfully developed. The method is rapid, sensitive and specific in use, showing potential diagnostic application value for schistosomiasis japonica.

    Evaluation of the intervention effect of comprehensive prevention and control measures on enterobiasis among children in Nanjing City
    HE Yisha, YANG Peicai, YIN Weigang, XIE Chaoyong
    2024, 42(4):  487-495.  doi:10.12140/j.issn.1000-7423.2024.04.010
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    Objective To evaluate the intervention effect of comprehensive prevention and control measures on enterobiasis among children so as to provide a informative basis for formulating and adjusting the prevention and control strategies. Methods Using random cluster sampling method, one kindergarten was selected from each of the 12 districts in Nanjing to conduct a survey on the infection of Enterobius vermicularis among all children in September to October 2022. Based on the survey results, four kindergartens with higher infection rates and similar environment and scale were selected from both urban and suburban areas, respectively. All children from 8 kindergartens, one guardian living with each child and the class teacher of each class were enrolled in the survey. Among them, two kindergartens in the urban area and two in the suburbs were assigned as the experimental group implemnting comprehensive control measures, while the remaining four kindergartens as the control group carring out traditional control measures; both groups conducted one-year intervention from November 2022 to November 2023. For the experimental group, the intervention measres comprised of symptom surveillance (on children’s suspected symptoms of Enterbobius infection), companion surveillance (sampling test for positive children’s family members living together with, classmates and playmates), and environmental surveillance (using transparent adhesive tape pasting method to collect environmental samples from positive children’s families and classes), as well as health education with three-dimension of “knowledge, attitude, and practice” as entering point to perform educational intervention jointly with multiple sectors including families, schools, and communities. The intervention measures for the control group were deworming for positive children only and health education delivered to the children and their family members. The After completion of the intervention study, anal swab method with transparent adhesive tape was used to examine the E. vermicularis infection rate in the children of two groups, respectively. Before and after the inervention, questionnaire survey on E. vermicularis control related knowledge was delivered to one class teacher of each class and one guardian living with each child, respectively. Results Before the intervention, the E. vermicularis infection rates in the experimental group and the control group were 1.5% (13/885) and 1.4% (12/886), respectively, showing no significant difference between the two groups (χ2 = 0.042, P > 0.05). A total of 234 children with suspected symptoms were found during symptom monitoring in the experimental group, among them, anal itching proportion was found the highes (34.62%, 81/234), followed by sleeping teeth grinding symptom (20.5%, 48/234). A total of 8 positive cases were detected, of which symptoms of anal itching accounted for the highest (5/8), followed by symptoms of sleeping teeth grinding (2/8). In companion surveillance, four positives were detected from positive children’s family members and two positives from their classmates, repectively. In environmental surveillance, four positives were found from plush toys samples, and two positive samples of blankets, sheets, and sofas were detected in the positive children’s family environment, respectively, and one positive sample was found from the desk and chair of their classroom, respectively. After one-year intervention, the infection rate in the experimental group decreased from 1.5% (13/885) before intervention to 0.1% (1/885) (χ2 = 10.368, P < 0.05), which was significantly lower than that in the control group after intervention (0.9%,8/886) (χ2 = 4.014, P < 0.05). The questionnaire showed that prior to the intervention, there was no significant difference in positive response rate towards children’s hygiene behaviour habits, and parents’ and teachers’ awareness points regarding to enterobiasis “knowledge, attitude, and practice” between the two the groups (all P > 0.05). The correct response rates to the questions “whether washing hands before and after meals, habits of trimming nails, biting fingers and biting toys”(89.9%, 796/885; 88.9%, 787/885; 85.8%, 759/885; 86.8%, 768/885) and correct hygiene habits rates of “whether have a separate towel, frequency of weekly showers, and frequency of monthly bedding drying” (89.2%, 789/885; 85.7%, 758/885; 78.9%, 698/885) in the experimental group were significantly higher than those in the control group (61.7%, 547/886; 71.2%, 631/886; 56.2%, 498/886; 59.8%, 530/886; 78.8%, 698/886; 78.8%, 698/886; 68.6%, 608/886) after intervention (χ2 = 192.194, 86.989, 187.741, 164.402, 35.371, 14.285, 24.010; all P < 0.05). After intervention, the positive response rates to all questions towards enterobiasis “knowledge, attitude, and practice” the experimental group were significantly higher those in the control group (all P < 0.05). In post-intervention, the positive response to all questions towards awareness of enteroviasis in teachers were all significantly higher than those in the control group (all P < 0.05). Conclusion Implementation of comprehensive prevention and control measures effectively reduced E. vermicularis infection rate in children, achieved timely interruption and precise control, meanwhile, raised parents’ and teachers’ health awareness and cognitive level on enterobiasis, and promoted changes of their attitudes and behaviours, driving children to develop good hygiene habits, and leading to effectively prevent and interrupt the transmission and spread of enterobiasis.

    Clinical and endoscopic characteristics of 10 cases of amoebic colitis
    XIE Qiao, LI Jun, DONG Lifeng
    2024, 42(4):  496-501.  doi:10.12140/j.issn.1000-7423.2024.04.011
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    Objective To analyze the clinical and endoscopic characteristics of 10 cases of amoebic colitis. Methods Data on 10 cases of amoebic colitis diagnosed in a tertiary hospital in Beijing from May 2021 to May 2024 was collected, including clinical manifestations, blood routine, stool routine, liver and kidney function, endoscopic mucosal features of colonic lesions, pathological features, treatment and prognosis, to summarize the clinical and endoscopic characteristics of amoebic colitis. Results All 10 patients were male, with an average age of (37.8 ± 13.1) years, ranging from 28 to 70 years among them, 4 cases had a history of sexually transmitted disease [3 cases of human immunodeficiency virus (HIV) infection, 1 case of syphilis], 1 case had a history of hepatitis C, and 1 elderly patient had a history of hypertension. Clinical symptoms include changes in defecation habits and stool shape, manifested as diarrhea and bloody stools, with 8 patients experiencing abdominal pain. All cases had mild tenderness in the abdomen. Blood routine and lymphocyte count were both normal, with only one patient having an increased proportion of eosinophils (13.4%). No amoebic cysts were found in the stool routine, and occult blood was positive in the stool. Eight patients had positive red blood cells and white blood cells in the stool, while two patients had negative results for both. Renal function was normal, and 9 patients had normal liver function and bilirubin levels. Only one patient with amoebic liver abscess had elevated bilirubin levels (total bilirubin 50 μmol/L, direct bilirubin 37.5 μmol/L). The endoscopic examination showed that the lesions in 10 patients mainly affected the ileocecal, ascending colon, and rectum segments, all presenting as scattered and irregular map-like ulcers, numbered in dozens and sized 5-30 mm in diameter. The surface of the ulcer was covered with a yellow or white coating, and the white coating overflows from the ulcer surface. The surrounding mucosa of the ulcer was congested, edematous and bloody, and the intestinal mucosa between the ulcers was normal. Among them, three cases with HIV-coinfection had larger ulcer areas. The hematoxylin-eosin staining showed that all 10 patients’ intestinal mucosal biopsy sections displayed inflammatory granulation tissue, a large amount of eosinophil and neutrophil infiltration in stroma, and amoeba trophozoites in inflammatory necrotic tissue and on mucosal surface. Among the ten patients, nine were given metronidazole (750 mg each time, 3 times a day) and diloxanide (500 mg each time, 3 times a day) orally for 10 days. One patient with amoebic colitis complicated with liver abscess was treated with intravenous injection of metronidazole (500 mg each time, once every 8 hours) for 14 days and then switched to oral metronidazole (750 mg each time, three times a day) for 10 days. After treatment, clinical symptoms such as abdominal pain, diarrhea, bloody stools, and purulent stools were significantly relieved in 10 patients. Among them, 6 patients had no abnormalities in routine stool examination and occult blood, and 2 patients had normal colon mucosa lesions on colonoscopy. Conclusion Amoebic colitis is more common in males, often seen in those with complicated sexually transmitted diseases. Clinical symptoms often include changes in bowel habits and hemafecia. Under endoscopy, the lessions mainly involve the ileocecal region, ascending colon, and rectum, presenting as ulcers of varying sizes. Diagnosis could be confirmed by finding of amoeba trophozoites in pathological biopsy.

    Analysis on DNA methylation levels in mosquitoes of the Culex pipiens complex
    GUO Sihan, HUANG Xin’an, XU Hanli, LI Chunxiao, LIU Kangkang, XING Dan, ZHAO Teng
    2024, 42(4):  502-511.  doi:10.12140/j.issn.1000-7423.2024.04.012
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    Objective To analyze and compare the DNA methylation levels of three subspecies of Culex pipiens complex, including Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus, and the DNA methylation levels of Cx. p. quinquefasciatus before and after blood-feeding. Methods Mosquitoes of the 3 subspecies were collected at 5 days post-feathering without blood-feeding and Cx. p. quinquefasciatus were collected at 3 days after blood-feeding. DNA was extracted and sonicated into fragments of approximately 250 bp. The fragmented DNA was sequenced, and the data were aligned with the reference genome sequence of Cx. p. quinquefasciatus (Taxonomy ID: 7176). Methylation levels of the 3 subspecies were analyzed at the genomic, chromosomal and elemental levels (the methylation level above 3% was considered hypermethylated). The differences in methylation levels among the 3 subspecies mosquitoes without blood-feeding, and in Cx. p. quinquefasciatus before and after blood-feeding were compared. The sites with P < 0.001 and absolute value of methylation difference > 5 were identified as differentially methylated sites. The regions with Q < 0.05 and the absolute value of methylation difference > 3 were identified as differentially methylated regions (DMR). The genes with the nearest transcription start site (TSS) to DMRs were identified as DMR-associated genes, which were subjected to gene ontology (GO) enrichment analysis. Results The genome-wide methylation levels of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus were 0.454%-0.672%, 0.491%-0.649% and 0.499%-0.655%, respectively, all were below 3%. The methylation levels of CHH of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus were 0.631%, 0.618% and 0.624%, respectively, which were higher than CHG (0.567%, 0.559%, 0.559%) (t = 7.14, 83.43, 6.87, all P < 0.05) and CG/CpG (0.508%, 0.505%, 0.505%) (t = 10.59, 12.52, 13.33, all P < 0.05). There were 56 hypermethylated sites and 11 hypermethylated regions present among all 3 subspecies. No significant differences were found among the 3 subspecies (F = 0.07, P > 0.05) at genome-wide methylation levels. No significant differences were found in methylation levels of chromosome 1 (.568%, 0.562%, 0.565%), 2 (0.573%, 0.564%, 0.566%) and 3 (0.575%, 0.566%, 0.569%) among the 3 subspecies at the chromosome level (F = 0.05, 0.11, 0.13, all P < 0.05) nor in the methylation levels of promoters (0.567%, 0.552%, 0.556%), exons (0.562%, 0.556%, 0.558%), introns (0.561%, 0.550%, 0.555%) and TSS (0.579%, 0.506%, 0.621%) among the 3 subspecies at the chromosomal elements level (F = 0.37, 0.06, 0.06, 0.16, all P > 0.05). There were 178 differentially methylated sites and 4 DMRs between Cx. p. pallens and Cx. p. molestus; 209 differentially methylated sites and 8 DMRs between Cx. p. pallens and Cx. p. quinquefasciatus; and 215 differentially methylated sites and 11 DMRs between Cx. p. molestus and Cx. p. quinquefasciatus. GO enrichment analysis revealed that the DMR-associated genes were mainly enriched in the biological processes with response to radiation, light stimuli and abiotic stimuli. The genome-wide methylation levels of Cx. p. quinquefasciatus slightly increased from 0.602% before blood-feeding to 0.617% after blood-feeding, without statistically significant differences (t = 1.21, P > 0.05). The methylation levels of chromosome 1, 2 and 3 in Cx. p. quinquefasciatus were 0.569%, 0.569% and 0.572% before blood-feeding, and were 0.596%, 0.597% and 0.600% after blood-feeding. There were no statistically significant differences before and after blood-feeding (t = 1.31, 1.33, 1.30, all P > 0.05). The methylation levels of the promoters, exons, introns and TSS in Cx. p. quinquefasciatus before blood-feeding were 0.557%, 0.561%, 0.560%, 0.552%, and were 0.585%, 0.584%, 0.584%, 0.594% after blood-feeding, respectively. There were no statistically significant differences before and after blood-feeding (t = 1.48, 1.35, 1.20, 1.69, all P > 0.05). There were 6 DMRs in Cx. p. quinquefasciatus between before and after blood-feeding. GO enrichment analysis showed that the DMR-associated genes were mainly enriched in endosomes and vesicles in cell components, and protein binding or small GTPases binding in molecular functions. Conclusion The genome-wide methylation levels of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus are relatively low. The DMR-associated genes are mainly related to biological processes that respond to abiotic stimuli. The methylation level of Cx. p. quinquefasciatus slightly increases after blood-feeding, and the DMR-associated genes before and after blood-feeding are mainly involved in protein binding.

    Cryptosporidium infection in rodents in China
    ZHOU Shanshan, JIANG Yanyan, CAO Jianping
    2024, 42(4):  512-520.  doi:10.12140/j.issn.1000-7423.2024.04.013
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    Cryptosporidium spp. are zoonotic intestinal protozoan with a wide distribution around the world.Cryptosporidiosis is mainly transmitted by fecal-oral route. Rodents are important hosts of Cryptosporidium spp. and in close contact with humans, making them one of the potential sources of Cryptosporidium infections in humans. Research data showed that 23 provinces had conducted surveys on Cryptosporidium infections in rodents in China, and there were differences in Cryptosporidium infections in rodents from different sources and uses. This paper summarized the distribution of species/genotypes and subtypes of Cryptosporidium infection in rodents from different regions, sources and uses in China, and analyses the effects of Cryptosporidium infections in rodents on other animals as well as the surrounding environment. Considering that Cryptosporidium spp. detected in rodents have also been detected in humans, other animals and their surroundings, the role of rodents in the transmission of Cryptosporidium spp. at the human-animal-environment interface cannot be ignored.

    STANDARD INTERPRETATION
    Interpretation of the Criteria for Detection of Malaria Parasite Nucleic Acid by Multiplex PCR Methods
    WANG Zhenyu, JIANG Li, YU Qing, ZHANG Yaoguang, WU Huanyu, CHEN Jian, ZHU Min
    2024, 42(4):  521-524.  doi:10.12140/j.issn.1000-7423.2024.04.014
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    The Criteria for Detection of Malaria Parasite Nucleic Acid by Multiplex PCR Methods (T/SPMA 004-2023) (referred to as the Criteria) was compiled following the Social Organization Standardization-Part 1: Guidelines for Good Practice (GB/T 20004.1-2016) and Standardization Working Guidelines (GB/T 1.1-2009). The Criteria is composed of seven chapters, including “the range of application” “normative reference documents” “terms and definitions” “instruments and equipment” “reagent and materials” “detection steps” and “waste disposal and pollution prevention measures”. One informative appendix (technical principle) and four normative appendices (two detection methods, reagent preparation and sample preparation). The Criteria was issued by Shanghai Preventive Medicine Association through No.34 of SJPM in 2023. The Criteria provides for the technical reference for normative operations and the quality of sample testing in disease control institutions and medical institutions. And the Criteria makes up for the deficiency of specific operating steps in nucleic acid detection of Criteria for Diagnosis of Malaria (WS 259-2015).

    SHORT COMMUNICATIONS
    Epidemiological characteristics of imported malaria in Shenyang, Liaoning Province from 2013 to 2022
    ZHANG Zhaoyu, JI Xingyu, WEN Lihai, WANG Caohaowei, JI Haitao, CHEN Huijie
    2024, 42(4):  525-528.  doi:10.12140/j.issn.1000-7423.2024.04.015
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    To understand the epidemiological characteristics of imported malaria in Shenyang, data on imported malaria cases and case investigations reported in Shenyang from 2013 to 2022 were collected from the National Diseases Reporting Information System and analyzed the type, population, time and source of imported malaria cases in Shenyang using descriptive epidemiological methods. A total of 176 imported malaria cases were reported in Shenyang from 2013 to 2022. The number of reported cases was the highest in 2016 and 2018, both at 25 cases (14.2%), while it was the lowest in 2022, at 5 cases (2.8%), with a decreasing trend. The majority of the reported cases were falciparum malaria (152 cases), accounting for 86.4%. Ovale malaria, vivax malaria and malariae malaria accounted for 7.4% (13/176), 4.5% (8/176) and 1.7% (3/176) respectively. There were 168 reported cases among males (accounting for 95.5%), and 8 reported cases among females (accounting for 4.6%), with a male-to-female ratio of 21∶1. The age of the reported cases was mainly in the age groups of 40-49 years (60 cases, 34.1%). Cases were reported throughout the year from 2013 to 2022, with the highest number of cases reported in September (12 cases, 11.4%). There was no obvious seasonal pattern. The sources of reported cases involved 28 countries from 3 continents, mainly from African countries, accounting for 95.6% (168/176). It suggested Shenyang should continue to strengthen the monitoring and case management of imported malaria, as well as enhancing the health education for entry-exit personnel, so as to prevent the re-transmission of imported malaria and consolidate the achievements of malaria elimination.

    Characteristics and case diagnosis analysis of imported malaria in Xuchang City, Henan Province from 2017 to 2023
    MA Yingying, PENG Jinhua, WU Ning, ZHANG Dongmin, ZHENG Huiping
    2024, 42(4):  529-532.  doi:10.12140/j.issn.1000-7423.2024.04.016
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    The data on imported malaria and epidemiological investigation of individual cases reported in Xuchang City, Henan Province, were collected from the Infectious Disease Surveillance Reporting and Management System, and the Parasitic Disease Prevention and Control Information Management System from 2017 to 2023. The epidemiological characteristics and case diagnosis were analyzed. There were 65 malaria cases reported in Xuchang City, including 48 Plasmodium falciparum infections (73.8%), 10 P. ovale infections (15.4%), 2 P. malariae infections (3.1%), 3 P. vivax infections (4.6%) and 2 mixed infections (3.1%). The male-to-female ratio was 20.7∶1. The cases were mainly reported by districts of Jian’an (30.8%, 20/65), Yuzhou (26.2%, 17/65) and Weidu (18.5%, 12/65), and there was no apparent seasonal variation in distribution. The time interval P50 between the onset and diagnosis of the cases was 2 days (P25, P75: 1 d, 4 d). The median time interval from onset to diagnosis for cases with and without a history of malaria was 2 days (1, 4), with no statistically significant difference (Z = -0.636, P > 0.05). The malaria diagnosis accuracy rate within 3 days after medical consultation was 83.1% (54/65). The proportion of malaria cases reported by provincial, municipal, and county (district) medical facilities in Xuchang City from 2017 to 2023 was 7.7% (5/65), 50.8% (33/65), and 41.5% (27/65), respectively. The proportion of initial diagnosis medical facilities at the provincial, municipal, county (district), and rural levels was 7.7% (5/65), 29.2% (19/65), 29.2% (19/65), and 33.9% (22/65), respectively (χ2 = 7.999, P < 0.05) and their malaria diagnosis accuracy rates were 5/5, 17/19, 16/19, and 0 (0/22), respectively (χ2 = 47.228, P < 0.01). The reported cases were all imported cases, mainly from Nigeria (20.0%, 13/65), the Democratic Republic of Congo (15.4%, 10/65), and Guinea (9.2%, 6/65) in Africa.

    Analysis of the epidemic characteristics of imported malaria in Longgang District, Shenzhen City, 2018-2023
    GAN Liqin, LI Yuan, LIU Wuyi, LIANG Xiangsheng, HUANG Dana, GAO Shitong
    2024, 42(4):  533-536.  doi:10.12140/j.issn.1000-7423.2024.04.017
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    In order to understand the epidemiological characteristics of imported malaria after malaria elimination in Longgang District of Shenzhen City and analyze the effect of epidemic surveillance and response, the surveillance data of malaria in Longgang District from 2018 to 2023 were collected from the National Information System for Infectious Diseases Surveillance, National Information System for Parasitic Diseases Control and Prevention, as well as medical institutions and annual reports. The vehicle monitoring was carried out. Descriptive epidemiological methods were used to analyze the data, such as the number of cases, parasite species, infection sources, clinical symptoms, and surveillance data. The results showed that 173 imported malaria cases were reported in Longgang District from 2018 to 2023, including 144 cases of Plasmodium falciparum infection (83.2%), 13 cases of P. ovale infection(7.5%), 11 cases of P. vivax infection (6.4%), 1 case of P. malariae infection (0.6%), and 4 cases of mixed infection with P. falciparum and P. ovale (2.3%). The main infection sources were Africa (169 cases, 97.7%) and Southeast Asia (4 cases, 2.3%). There were 7 outpatients (4.0%) and 166 inpatients (96.0%), including 1 severe case (0.6%). The number of imported cases peaked in July, August and October (21, 51 and 20 cases), and February saw the fewest cases (2 cases). Most of the reported cases were young and middle-aged males, with a male-to-female ratio of 13.4 ∶ 1. From 2018 to 2023, the positive rate of Plasmodium blood smears in Longgang District was 6.0‰ (173/29 025), and the average time from initial diagnosis to definitive diagnosis decreased from 2.32 d during 2018-2020 to 0.58 d during 2021-2023 (t = 2.102, P < 0.05). The correct rate of initial diagnosis was 96.4% (135/140) at the city level, 69.0% (20/29) at the county level, and 0/4 at community health service centres and other medical institutions (Fisher’s exact test, P < 0.01). The correct rates of microscopic examinations for P. falciparum, P. malariae, P. vivax, P. ovale and mixed infections were 88.9% (128/144), 1/1, 11/11, 8/13 and 2/4, the difference was statistically significant (Fisher’s exact test, P < 0.01). All the cases were reported within 1 day, and after finishing laboratory review, epidemiological investigation and case report within 3 days, all the epidemic spots were disposed of within 7 days. None of the cases gave rise to secondary cases. Anopheles mosquitoes were not found in Longgang from 2018 to 2023. Longgang District is under great pressure for the prevention and control of imported malaria after malaria elimination. The malaria surveillance and response system in medical and disease control institutions operates well. However, the capacity for malaria diagnosis, treatment, and laboratory testing among the medical staff and institutions should be further improved.

    Analysis on surveillance of soil-transmitted nematode infections in Shandong Province from 2021 to 2022
    WANG Longjiang, XU Yan, SUN Hui, LI Yuejin, BU Cancan, LV Wenxiang, ZHANG Benguang, KONG Xiangli, YAN Ge, WANG Yongbin
    2024, 42(4):  537-541.  doi:10.12140/j.issn.1000-7423.2024.04.018
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    In order to understand the current situation of soil-transmitted nematode infections in humans in Shandong Province and provide the scientific basis for optimizing prevention and control strategies, from 2021 to 2022, 27 counties in Shandong Province were randomly selected as surveillance sites. Each surveillance site was divided into five regions (east, west, south, north, and central) based on geography, and one administrative village from one township was randomly selected in each region. A total of 200 permanent residents with an age of over 3 years were taken as the surveillance object from each village by cluster sampling method. Basic information and the fecal samples were collected, and the worm eggs were examined in duplicates using the modified Kato-Katz thick smear method (one sample, two slide-readings) and counted. Hookworm species were identified using the tube filter paper culture technique. Children aged 3-9 years old were tested for Enterobius termicularis by anal transparent tape. A database was established, and SPSS 26.0 software was used to perform the statistical analysis. The infection rate and Infectivity were calculated. The infection rates were analyzed using the Chi-square test. A total of 29 622 people were detected in the province in 2021-2022, and the total infection rate of soil-transmitted nematode was 0.55% (162/29 622). The infection rates of hookworm, Ascaris lumbricoides and Trichuris trichiura were 0.02% (7/29 622), 0.02% (6/29 622) and 0.51% (152/29 622), respectively. The hookworm samples were all identified as Ancylostoma duodenale. Among the 152 trichuriasis patients, 2 were severely infected, 6 were moderately infected, and the rest were slightly infected. Six A. lumbricoides infections and seven hookworm infections were mild infections. The prevalence of soil-transmitted nematode was 0.97% (118/12 208), 0.84% (43/5 117), 0.01% (1/7 140), 0 (0/5 157) in southern, eastern, northwestern and central Shandong. The infection rate of soil-transmitted nematode was 0.55% (79/14 362) in males and 0.54% (83/15 260) in females (χ2 = 0.005, P > 0.05). The age group of 60-99 had the highest infection rate among all age groups accounting for 1.11% (107/9 612) (χ2 = 86.274, P > 0.05). Farmers had the highest infection rate among different occupational groups, accounting for 0.78% (148/18 863) (χ2 = 54.078, P < 0.05). The illiterate or semi-illiterate group had the highest infection rate among different education levels, accounting for 2.43% (60/2 468) (χ2 = 175.458, P < 0.05). A total of 2 584 children were examined, and the infection rate of E. vermicularis was 1.16% (30/2 584). The infection rate in boys and girls was 1.60% (22/1 375), 0.66% (8/1 209) (χ2 = 4.936, P < 0.05). The highest rate was found in 7-year-old children, accounting for 4.50% (15/333) (χ2 = 39.798, P < 0.05). The distribution of soil-transmitted nematode in Shandong Province was unbalanced among regions and populations, which indicated that the prevention and control of soil-transmitted nematode should be strengthened in key areas and carry out effective health education for key populations to reduce the infection rate continuously.

    Epidemiological trend of hookworm infection in Jiangxi Province
    ZHOU Yuehong, JIN Zhimei, QIU Tingting, LI Peng, LAN Weiming, CHEN Zhe, GE Jun
    2024, 42(4):  542-546.  doi:10.12140/j.issn.1000-7423.2024.04.019
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    To analyze the epidemic trend and current situation of hookworm disease in Jiangxi Province, all for providing a basis for formulating countermeasures of hookworm disease. The survey data of hookworm disease in Jiangxi Province in three national sampling surveys of human parasitic diseases (1989, 2002 and 2015) and the surveillance data of hookworm infection in the national monitoring sites of soil nematode disease in Jiangxi Province from 2016 to 2020 were analyzed, respectively. The Kato-katz smear method was used in the survey and surveillance to examine the hookworm infection, and the test-tube filter paper culture method was used to identify hookworm species. The results showed that the infection rate of hookworm in Jiangxi were 17.63% (9 176/52 042) in 1989, 7.39% (1 490/20 154) in 2002 and 4.63% (1 094/23 606) in 2015 with the downtrend as a whole (χ2 = 3 112.64, P < 0.05). The infection rates in female in 1989 and 2015 (18.68% and 5.59%) were higher than that in male (16.60% and 3.88%) (χ2 = 38.71, 28.85, both P < 0.05). As for the age distribution of infection in 3 national surveys, the infection rate in age group of 50-59 in 1989 was the highest (27.33%), and the rates in ≥ 60 age group in 2002 and 2015 were the highest accordingly (12.37% and 10.06%). The dominant species of hookworm in 1989 was Ancylostoma duodenale, while Necator americanus becoming dominant in 2002 and 2015. The infection rates of hookworm from 2016 to 2020 in Jiangxi were 1.04% (116/11 126), 1.09% (122/11 203), 0.63% (70/11 183), 1.00% (112/11 197) and 0.62% (70/11 240), respectively and continued to decline (χ2 = 44.85, P < 0.01). In the gender distribution, the infection rate of female (0.97%, 269/27 718) was still higher that of male (0.78%, 221/28 231) (χ2 = 5.98, P < 0.05). In the age distribution, the infection rate in ≥ 60 age group was still the highest (1.51%, 235/15 594). The analysis results indicated that the infection rate of hookworm shows a continuous decline trend, and maintains a low epidemic state in Jiangxi. Rural women and high age groups are the focus of hookworm control.

    CASE REPORTS
    A case of imported schistosomiasis haematobia from a foreigner
    LI Rui, YU Yingying, ZHUO Wanjun, YANG Shouwang, CHEN Linbi, HUANG Yishu
    2024, 42(4):  547-549.  doi:10.12140/j.issn.1000-7423.2024.04.020
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    A 19-year-old male patient of Nigerian nationality, an international student of a university in Wenzhou, came to Wenzhou International Travel Healthcare Centre for entry medical examination on October 23, 2023, and reported no symptoms of discomfort. The blood routine test showed an increased percentage of eosinophils (15.9%), and the serum immunoglobulin test showed elevated levels of IgM antibodies (2.37 g/L). The urine routine test showed positive occult blood (2 +), positive protein (1 +), and elevated counts of both red blood cells (360 RBCs per μl) and white blood cells (255 WBCs per μl). Schistosoma haematobium eggs were found in the urine sediment by microscopy, and miracidia were hatched from the eggs. Nucleic acid testing of the urine sediment confirmed a positive result for S. haematobium. The B-ultrasonic showed slight thickening of the bladder wall. Epidemiological investigation suggested that the patient might have been exposed to contaminated water while swimming in lakes in Nigeria. Based on laboratory examination and epidemiological investigation, the patient was diagnosed as imported schistosomiasis haematobia. On October 27, the patient took orally praziquantel, 30 mg/(kg·d), 3 times a day for 2 consecutive days. A follow-up examination on November 13 revealed no eggs in the urine sediment.

    A case of Fasciola hepatica infection combined with Pseudomonas aeruginosa in a child
    BAI Houxi, YANG Xiaotao, LUO Yonghan, WANG Yanchun
    2024, 42(4):  550-553.  doi:10.12140/j.issn.1000-7423.2024.04.021
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    The patient was a 13-year-old male from Yun County, Lincang City, Yunnan province. He went to the local city hospital on October 20, 2023, for treatment due to “Fever with right upper abdominal pain for over 2 days”. The abdominal CT showed uneven enhancement of the liver parenchyma with multiple patches of slight hypoenhancement, suggesting an infectious lesion. He was given ceftriaxone with other anti-infection treatments (dosage course of treatment was unknown) and praziquantel against helminth infections, but the symptoms did not improve. He was transferred to Kunming Children’s Hospital on November 9 for inpatient treatment. The physical examination showed a slight bulge below the xiphoid, pressure pain in the right epigastric region, a mass about 3 cm × 2.5 cm in size palpable at the outer edge of the rectus abdominis muscle on the right side, and the liver palpable 4 cm below the right costal margin and 5 cm below the xiphoid. Routine blood tests showed an elevated eosinophil count (1.02 × 109/L) and elevated C-reactive protein (CRP) (68.54 mg/L). The other blood tests showed elevated alanine aminotransferase (90 U/L), aspartate aminotransferase (51 U/L), γ-glutamyltransferase (296 U/L), alkaline phosphatase (794 U/L), and immunoglobulin G (34.55 g/L), with accelerated sedimentation rate (67 mm/h). The blood cultures were negative. No eggs were found in stool under microscopy. Abdominal CT scan and enhancement showed multiple hypodense foci in the liver parenchyma, intrahepatic bile duct dilatation and possible cholangitis. The result of enzyme-linked immunosorbent assays for serum antibody against Fasciola hepatica was positive. Five sequences of F. hepatica and eight sequences of Pseudomonas aeruginosa were detected in the venous blood by metagenominext-generation sequencing, and no sequences of other pathogens were detected, such as fungi and viruses. The patient lived in a rural area and had a history of consuming cold watercress and drinking unboiled water. Epidemiological investigation, physical examination and relevant auxiliary test results led to the final diagnosis of F. hepatica combined with P. aeruginosa infection. Triclabendazole (500 mg/d once daily) was given for 2 days for deworming and meropenem [60 mg/(kg•d), three times a day)] was given as anti-infection treatment for 2 weeks, and he was discharged from the hospital without further fever and abdominal pain. After 3 months of follow-up and reexamination after discharge, no abdominal mass was touched, and the blood eosinophil count was normal. The abdominal CT scan showed that most of the lesions were smaller than before.

    A case of imported angiostrongyliasis in Shandong Province
    LI Yuejin, YAN Yan, SHEN Yanmei, WEI Yanbin, XU Yan, WANG Longjiang, YAN Ge, GUO Yunhai, WANG Yongbin
    2024, 42(4):  554-556.  doi:10.12140/j.issn.1000-7423.2024.04.022
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    The patient is a 34-year-old male from Qingdao, Shandong, who works in the insurance industry. He experienced headaches for over a month and visited the Qingdao Branch of Qilu Hospital of Shandong University on May 31, 2023, due to “worsening headaches”. He was admitted for intracranial infection and treated with acyclovir, dexamethasone, and mannitol (specific doses not provided). On June 18, he was transferred to the Affiliated Hospital of Digestive Diseases of Shandong First Medical University. The patient reported travelling to Dali, Yunnan, on May 1, 2023, during which he consumed snail meat. After returning home, he began experiencing headaches, accompanied by general discomfort and chest tightness. Admitted for physical examination, he remained conscious, with significant headaches in an upright position, and had a normal diet and bowel movements. Blood routine tests showed elevated eosinophil count (0.86 × 109/L) and percentage (10.70%). A plain cranial CT scan revealed normal brain parenchyma density with no abnormal density shadows or space-occupying lesions. The ELISA results of cerebrospinal fluid and blood samples showed weakly positive IgG antibodies for Angiostrongylus cantonensis, while other parasite antibody were negative. He was diagnosed with angiostrongyliasis. He was treated with albendazole [20 mg/(kg·d), 3 times a day, continuous oral administration for 7 days] and dexamethasone [0.15 mg/(kg·d) intravenously for 3 days] for treatment. On June 28, the headache symptoms of the patient had disappeared, and his eosinophil count and percentage returned to normal values, and he was discharged. Two months later, a follow-up examination showed normal blood tests, negative IgG antibody tests for A. cantonensis, and no other discomfort.

    A case of visceral leishmaniasis related hemophagocytic syndrome
    YAN Dongning, MOU Xiaoli, CHEN Xiaojun
    2024, 42(4):  557-558.  doi:10.12140/j.issn.1000-7423.2024.04.023
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    A 60-year-old female patient from Longnan of Gansu Province, was admitted to a local county hospital on August 25, 2023, due to fever, fatigue and poor appetite. After completing relevant examinations, she was considered to have hematological diseases. After symptomatic treatments such as blood transfusion and anti-infection, the patient still had recurrent fever. On September 7, 2023, the patient was treated in the People’s Hospital of Gansu Province. Physical examination on admission showed that the spleen was 2 cm below the costal edge, tough and without tenderness. Blood routine showed that white blood cell count was 1.5 × 109/L (↓), red blood cell count was 3.8 × 1012/L (↑), hemoglobin was 68 g/L (↓), platelet count was 4.3 × 1010/L (↓), Ferritin was over 2 000 μg/L (↑), triglyceride was 2.4 mmol/L (↑), fibrinogen was 1.4 g/L (↓) and sluble CD25 was 1 202 U/ml (↑). The results of bone marrow cytological analysis showed that the special macrophages phagocytized living blood cells. Bone marrow smear microscopy showed that some of the macrophages had Leishmania donovani amastigotes in and around the cytoplasm. Combined with the epidemiological history, the patient had long lived in a mountainous area in Longnan of Gansu Province, which is the main endemic area of visceral leishmaniasis in China. The patient was diagnosed with visceral leishmaniasis related hemophagocytic syndrome. After treatment with gluconate (6 ml/d) and dexamethasone (15 ml/d) for one week, the patient had no fever. Reexamination on September 18 showed that white blood cell count was 4.5 × 109/L, red blood cell count was 4.3 × 1012/L, hemoglobin was 80 g/L, platelet count was 1.1 × 1011/L and fibrinogen was 1.51 g/L, all of which were higher than those at admission. Five months later, the blood test routine showed that the white blood cells, hemoglobin and fibrinogen returned to normal. Platelets were elevated, but still below the normal range. Abdominal ultrasound showed that the size of the liver and spleen were normal.