Table of Content

30 April 2024, Volume 42 Issue 2

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SPECIAL REPORT

Epidemiological characteristics of malaria in China, 2023

ZHANG Li, XIA Zhigui

2024, 42(2):  135-139.  doi:10.12140/j.issn.1000-7423.2024.02.001

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The individual malaria case investigation forms in 31 provinces/municipalities/autonomous regions (excluding Taiwan, Hong Kong and Macao) and Xinjiang Production and Construction Corps of China in 2023 were collected and sorted from “The Information System for Infectious Disease Surveillance”. The epidemiological characteristics were analyzed. In 2023, 2 488 malaria cases were reported, showing an increase of 194.4% compared to 2022 (845). Among them, there were 2 487 imported cases, 1 case of blood transfusion infection; 1 561 falciparum malaria cases (62.7%, 1 561/2 488), 615 vivax malaria (24.7%, 615/2 488), 234 ovale malaria (9.4%, 234/2 488), 66 malariae malaria (2.7%, 66/2 488) and 12 cases with mixed-infection (0.5%, 12/2 488). In the reported, 2 313 cases were of Chinese nationality (93.0%, 2 313/2 488) and 175 cases foreign nationality (7.0%, 175/2 488); the male-to-female ratio was 11.6∶1; the highest number of cases (29.1%, 725/2 488) was seen at the age group of 30-39 years showed. The malaria cases were reported from all the 31 provinces/municipalities/autonomous regions and Xinjiang Production and Construction Corps, with the top 5 provinces of Yunnan (398 cases), Henan (234 cases), Guangxi (195 cases), Shandong (178 cases) and Guangdong (174 cases) accumulately, a total of 1 179 cases (47.4%, 1 179/2 488) reported. Of the reported, 85 were severe cases (3.4%, 85/2 488) and 12 deaths (0.5%, 12/2 488). Although no local transmitted primary malaria cases have been reported in China for seven consecutive years, the risk of imported cases and re-transmission. It is imperative to continuousely strengthen the surveillance and response, find the cases timely and deliver standardized treatment, so as to reduce severe cases and death, preventing local re-transmission.

ORIGINAL ARTICLES

Epidemiological characteristics of imported malaria in Shandong Province in 2017—2022

XU Yan, WANG Longjiang, KONG Xiangli, LI Yuejin, BU Cancan, YAN Ge, ZHANG Benguang, WANG Yongbin

2024, 42(2):  140-146.  doi:10.12140/j.issn.1000-7423.2024.02.002

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Objective To understand the epidemiological characteristics of imported malaria in Shandong Province and provide scientific basis for the prevention of re-transmission of imported malaria. Methods Data of the imported malaria epidemiological situation and case investigation in Shandong Province from 2017 to 2022 were collected from the Infectious Disease Reporting Information Management System and the Parasitic Disease Prevention and Control Information Management System of China Disease Prevention and Control Information System. A database for analysis was established. SPSS 26.0 software was use to analyze the case classification, source of infection, epidemiological characteristics, and the diagnosis and treatment records of the malaria cases. The χ² test was used to compare the rates, and the nonparametric test was used to compare the time interval distribution. The spatial clustering of malaria cases was analyzed by scanning with SaTScan v10.1 software. Figures were generated using GraphPad Prism 8.3.0 software. Results From 2017 to 2022, a total of 820 imported malaria cases were reported in Shandong Province, all of which were imported from abroad. A total of 670 cases were reported before the corona virus disease 2019 （COVID-19） pandemic and 150 cases during the COVID-19 pandemic. All the 820 cases were laboratory-diagnosed. falciparum malaria, ovale malaria,vivax malaria and malariae malaria accounted for 69.5% (570/820), 19.0% (156/820), 7.3% (60/820) and 4.1% (34/820), respectively. There were no mixed infections and unclassified cases. The main source of the infections was Africa (95.9%, 786/820). All 16 cities in Shandong Province had cases reported, while 511 cases were reported in five cities, including Jining, Yantai, Weihai, Qingdao and Jinan, accounting for 62.3% (511/820). Spatial scanning analysis found 7 case clusters, among which 13 counties, such as Huancui in Weihai and Muping in Yantai were the primary clusters (relative risk = 3.98, P < 0.01). According to the city where the case was reported, 191 cases had been treated in a different region within the province, accounting for 23.3% (191/820). A total of 421 local cases (55.3%, 421/762) were reported by 18 medical facilities designated for malaria treatment in the province. The median time interval from onset of symptoms to initial diagnosis and from initial diagnosis to final diagnosis was 1 and 0 days, respectively. The proportion of patients who were initially diagnosed in the provincial, municipal, county, and below-county medical facilities was 9.9% (81/820), 36.1% (296/820), 31.5% (258/820) and 16.6% (136/820), respectively. The malaria diagnosis accuracy was 88.9% (72/81), 84.1% (249/296), 76.4% (197/258) and 5.2% (7/136), respectively, with 6.0% (49/820) of the patients initially diagnosed in other institutions, such as centres for disease control, inspection and quarantine organizations, and the malaria diagnosis accuracy was 87.8% (43/49). There was a significant difference in the malaria diagnosis accuracy in different institutions (χ² = 321.959, P < 0.01). A total of 74 severe malaria cases were reported, accounting for 9.0% (74/820), and 5 cases decesed. Conclusion There are high number of imported malaria cases in Shandong Province, which are widely distributed. The risk of re-transmission exists.

Epidemiological analysis of imported malaria in Shanghai Public Health Clinical Center from 2012 to 2022

WEN Jing, GUO Mingquan, ZHANG Bei, ZHANG Tengfei, PAN Shuai, SUN Danfeng, QI Weiqiang

2024, 42(2):  147-152.  doi:10.12140/j.issn.1000-7423.2024.02.003

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Objective To analyze the epidemiological characteristics of malaria cases reported in Shanghai Public Health Clinical Center to provide scientific basis for surveillance and prevention malaria of imported in the pos-elimination phase. Methods Data of the malaria cases from 2012 to 2022 were collected from the information management system of Shanghai Public Health Clinical Center and analyzed using descriptive statistical method on the infected species, source of infection, triple distribution, and clinical visits and diagnosis. Results The results showed that a total of 248 malaria cases were reported in Shanghai Public Health Clinical Center during 2012—2022, and all cases were imported from overseas. There were 228 patients of Chinese nationality (91.9%) and 20 patients were foreigners (8.1%). The most cases were Plasmodium falciparum infection (208 cases, 83.9%). The highest number of cases was seen in 2017. The infecting cases were mostly imported from African countries (229 cases, 92.3%) and others were from Asian (13 cases, 5.2%)， South American countries (1 case, 0.4%) and unkown area (5 cases, 2.0%). There were cases reported in every month, and there was no seasonal pattern on the number of imported cases. The regional distribution of imported cases showed that 126 cases were located out of Shanghai, 114 cases were in Shanghai. There were 8 cases unknown. Pudong New Area (24 cases, 21.1%), Minhang District (14 cases, 12.3%) and Songjiang District (11 cases, 9.6%) had the highest number of cases in Shanghai. The reported malaria cases comprised 234 males (94.4%) and 14 females (5.6%), with a male-to-female ratio of 17 ∶ 1. The age of the cases were mainly in the age groups of 20-49 years (203 cases, 81.9%). The occupational background of the cases was dominated by migrant workers (187 cases, 75.4%). The number of cases treated at non-Shanghai hospitals and Shanghai hospitals were 14 and 234 cases, respectively. The malaria successful diagnosis rate was 96.3% (154/160) in tertiary A-level hospital, and 81.1% (60/74) in lower medical institutions during 2012—2022 in Shanghai. According to the 248 malaria cases, the average time period from the onset of symptoms to initial examination was 4.5 days, with a median of 2 (0，4) d. Of the Shanghai cases, 189 cases (76.2%) had initial examination 3 days after the onset of symptoms, 51 cases (20.6%) had that 4-10 days after the onset of symptoms, and 8 cases (3.2%) had that 10 days after the onset of symptoms. There was a significant difference between the median time from onset of symptoms to initial examination of malaria (F = 6.393, P < 0.05). The mean time intervals from initial examination to diagnosis was 1.2 d, and the median was 1 (0, 2) d. Of these cases, 112 patients were diagnosed on the day of first examination; 223 cases were diagnosed within 3 days after the initial exminatio and 25 were diagnosed more than 3 days after the initial examination. There was no significant difference between the median time from first examination to diagnosis (F = 1.24, P > 0.05). Conclusion The malaria cases reported in Shanghai Public Health Clinical Center from 2012 to 2022 were all imported from abroad with the dominant species of P. falciparum. The infection source was mainly from African countries. It is urged that Shanghai should continuously strengthen the surveillance and management of imported malaria, enhance the capability of diagnosis and clinical care in medical institutions, and strengthen health education for outbound personnel to consolidate the achievements of malaria elimination.

Polymorphism of sulfadoxine-pyrimethemine resistant gene of Plasmodium falciparum in China-Myanmar border area from 2010 to 2018

YAN He, HUANG Fang, FENG Jun, YIN Jianhai, XIA Zhigui, CAO Jianping

2024, 42(2):  153-159.  doi:10.12140/j.issn.1000-7423.2024.02.004

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Objective To analyze the polymorphism of sulfadoxine-pyrimethemine (SP) resistant gene and resistance recovery mutation trend of Plasmodium falciparum in Yunnan China-Myanmar border area. Methods P. falciparum blood filter paper samples were collected from China-Myanmar border area in 2010 to 2018. The target gene fragments P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) were amplified by nest-PCR, of which the DNA sequences were aligned by Geneious Prime software. Microsoft Excel 2016 and GraphPad Pism 8.0.2 software were used to analyze the differences in mutation frequency and distribution of haplotype. Haploview software was used to analyze the linkage disequilibrium of gene 180 pfdhfr and 178 pfdhps, chi-square test was used to analyze the characteristics of single nucleotide polymorphisms at resistance-associated gene loci and the varying distribution of haplotypes in different years. Results A total of 190 P. falciparum blood filter samples were collected in China-Myanmar border area. Specifically, 180 pfdhfr and 178 pfdhps gene fragments were successfully amplified and sequenced. The mutated amino acid codon locus in pfdhfr 51, 59, 108 and 164 was changed as N51I, C59R, S108N, I164L; which in pfdhps 436, 437, 540 and 581 was changed as S436 A and C, A437G, K540E and N, A581G. The resistant site frequency in Pfdhfr N51I, C59R, S108N and I164L was respectively 57.8% (108/187), 90.1% (172/191), 93.3% (168/180), 59.6% (109/183), while in pfdhps S436A\C, A437G, K540E\N, A581G the resistant site frequency was respectively 54.7% (99/181), 86.5% (154/178), 84.9% (152/177) and 28.7% (51/178). The majority of pfdhfr genotypes were focused on triple and quadruple site mutations (51I59R108N/59R108N164L, 51I59R108N164L), with 44.9% (84/187) and 36.9% (69/187), respectively. The wild types of two genes were not detected in 2010. The triple and quadruple mutated haplotypes frequency (51I59R108N/59R108N164L, 51I59R108N164L) with 35.9% (23/64) and 37.5% (24/64) in 2011 was obviously less than in 2010 with 46.2% (30/65), 49.23% (32/65) and 53.4% (32/58), 22.4% (13/58) from the year 2014 to 2018 (P < 0.05). The pfdhps triple mutated haplotypes distribution frequency 62.1% (36/58) (436A\C437G540E\N, 437G540E\N/581G) after the year 2014 was significantly less than 82.3% (51/62) in 2010 and 78.7% (48/61) in 2011 (P < 0.05). The Linkage disequilibrium analysis between the two loci C59R and S108N in pfdhfr showed a strong association with D’ = 1 and r² = 0.8， S436A and A581G in pfdhps gene showed a strong association with D’ = 1 and r² = 0.6. Meanwhile, S108N in pfdhfr and K540E in pfdhps were associated with D’ = 0.8, r² = 0.2. Conclusion It demonstrated that P. falciparum resistant gene pfdhfr and pfdhps presented with mutated haplotype as the dominant gene in Yunnan, China-Myanmar border area, furthermore, its mutation frequency may reduce as the medication discontinuation time for the host extended.

Activation of astrocytes and neuronal damage induced by the inflammatory microenvironment of cerebral malaria

TONG Guodong, ZHU Qinghao, WANG Jun, LIU Xiaoran, SHEN Yan, LIANG Jiao, LI Yinghui, HUANG Yuxiao, WANG Yi, ZHAO Ya

2024, 42(2):  160-168.  doi:10.12140/j.issn.1000-7423.2024.02.005

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Objective To explore the activation of astrocytes induced by the perivascular inflammatory microenvironment during the pathogenesis of the brain during the pathogenesis process of cerebral malaria and its mechanism, as well as its impact on neuronal damage. Methods C57BL/6 male mice aged 4-5 weeks were intraperitoneally inoculated with 5 × 10⁶ red blood cells infected with the ANKA strain of Plasmodium berghei, which would be died 7-10 d later and served as an experimental cerebral malaria model. Newborn suckling mice aged 3-5 d were euthanized to isolate primary astrocytes from the cerebral cortex; while the other suckling mince were euthanized within 24 hours for isolation of primary cortical neurons. The cell culture containing 20 ng/ml γ interferon (IFN-γ), 1 ng/ml tumor necrosis factor α (TNF-α) and mice red blood cells (pRBC) infected with malaria parasites were used to induce activation of astrocytes, and assigned as the activation group, while untreated resting-state cells were set as the control group. After cultivation for 24 h, total RNA was extracted by cell lysis and sequenced. Cluster analysis was performed to generate heatmaps, and representative genes were selected to draw violin diagram. ELISA was used to determine the expression level of CXCL10 in cell culture supernatant. The spleen CD8⁺ T cells of mice with cerebral malaria were separated and co-cultured with activated astrocytes to observe immune synapses and CXCL10 levels after co-incubation with CXCL10 antibodies by fluorescence microscopy, and CD80 and other molecular expression levels were detected using flow cytometry. Primary neurons were added into two sets of astrocyte culture supernatants and incubated in MAP2 antibody. Blank culture medium was set as the blank group, and lactate dehydrogenase (LDH) and CCK-8 assay kit were used to detect the degree of neuronal damage. Western blotting was used to detect the levels of STAT1, STAT3, and their phosphorylated molecules. The activation group was treated with STAT1 pathway inhibitor fludarabine, and the morphology of neurons was observed by immunofluorescence staining. Statistical analysis was conducted using PRISM Graph Pad 8.0 software, and pairwise comparisons were conducted using t-tests. Results The morphology of activated astrocytes changed from flat star-shaped to long spindle-shaped, and the relative content of glial fibrillary acidic protein (GFAP) was (1.36 ± 0.03), higher than that of the control group (1.00 ± 0.00) (t = 13.33, P < 0.01). The CXCL10 content in the supernatant of the activation group cell culture was (7.07 ± 0.81) ng/ml, which was higher than that of the control group (2.57 ± 0.28) ng/ml (t = 9.05, P < 0.01). Transcriptome analysis showed that antigen processing, presentation, and transcription levels of T cell chemokines were upregulated in the activation group (all P < 0.01), and the expression levels of CD80, CD86, and MHC Ⅰ were upregulated. The activation group co-incubated with CD8⁺ T cells to form "immune synapses", and the CD69 expression level of CD8⁺ T cells increased. The LDH results showed that the relative mortality rate of neurons in the activation group after supernatant stimulation was (50.2 ± 2.4)%, which was higher than that in the blank group (0% ) and the control group (0%) (t = 20.62, 20.62, both P < 0.01). The CCK-8 detection results showed that the relative activity of neurons in the activation group after supernatant stimulation was 0.52 ± 0.03, lower than that in the blank group (1.00 ± 0.00) and the control group (1.42 ± 0.06) (t = 18.92, 16.65, both P < 0.01). Western blotting results showed that relative expression level of neurons stimulated with activated astrocytes supernatant β-amyloid precursor protein (β-APP) was 0.44 ± 0.02, which was lower than that of the blank group (1.00 ± 0.00) and the control group (0.55 ± 0.02) (t = 37.28, 4.93, both P < 0.01). The ratio of pro apoptotic protein Bax to anti apoptotic protein Bcl-2 was 1.01 ± 0.07, and there was no statistically significant difference compared with the blank group (1.00 ± 0.00) and the control group (1.00 ± 0.06) (t = 0.31, 0.13, both P > 0.05). Western blotting results showed that the relative expression level of STAT1 and p-STAT1 protein in the cytoplasm of activation group were 3.40 ± 1.08 and 4.00 ± 0.82, both of which were higher than the control group (1) (t = 3.13, 5.13, both P < 0.05). The relative expression level of STAT3 and p-STAT3 protein in the cytoplasm of activation group were 1.00 ± 0.03 and 1.01 ± 0.05. There was no statistically significant difference compared to the control group (1) (t = 0.27, 0.52, both P > 0.05). The relative expression level of p-STAT1 protein in the nucleus of the activation group was 1.78 ± 0.21, higher than that of the control group (1) (t = 5.081, P < 0.01), and the relative expression level of p-STAT3 protein was 1.02 ± 0.02, with no statistically significant difference compared to the control group (1) (t = 1.38, all P > 0.05). The results of MAP2 immunofluorescence staining showed that the activation group supernatant could cause significant neuronal damage, which was alleviated after treatment with STAT1 inhibitors. Conclusion During the pathogenic process of cerebral malaria, the brain perivascular inflammatory microenvironment may induce production of neurotoxic astrocytes via the STAT1, and may cause death of neurons. This activated astrocytes could interact with activated CD8⁺ T cells, and further aggravate the damage of central nervous system.

Effect of local complement activation in hepatocytes on the development of Plasmodium in the infrared phase

TAN Nie, JIAO Shiming, DING Yan, ZHU Chengyu, XU Wenyue

2024, 42(2):  169-176.  doi:10.12140/j.issn.1000-7423.2024.02.006

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Objective To investigate the effect of hepatocyte local complement on the development of the liver-stage of Plasmodium parasites. Methods Hepa1-6 cells and HepG2-CD81 cells were collected, lysed, and RNA was extracted, respectively. Reverse transcription PCR amplified C3, C3aR1, C5, C5aR1 gene. Hepa1-6 cells and HepG2-CD81 cells suspension were lysed with protease and phosphatase, respectively. The protein concentration was measured using the bicinchoninic acid (BCA) assay and the expression of complement and receptor in hepatocytes was measured using Western blotting. Kunming mice fed with the BY265-RFP strain of P. yoelii expressing red fluorescence and the ANKA strain of P. berghei were fed with Anopheles stephensi for blood sucking. After 17-19 days, anatomically separated salivary glands from An. stephensi mosquitoes and added 50 000 sporozites into 24-well plate containing 10⁵ HepG2-CD81 cells per well for co-incubation, set an additional group that the cells were incubated with C5aR antagonist (40 nnmol/L, 500 μl/well）3 h after co-incubation. Cells were fixed with 4% paraformaldehyde and blocked, the non permeable group was incubated overnight at 4 ℃ with primary C3a rabbit anti human IgG antibody (1∶500) or rC5a rabbit anti human IgG antibody (1∶500), followed by 1 hour of incubation with green Dylight 488 fluorescent labeled goat anti rabbit IgG secondary antibody (1∶400), and 5 minutes of incubation with DAPI staining solution; added a primary antibody UIS4 goat polyclonal antibody (1∶500) to the permeable group and incubated overnight at 4 ℃, then added green IFKine^TM fluorescent labeled donkey anti rabbit IgG secondary antibody (1∶400) and incubated for 1 h, added CD88 rabbit polyclonal antibody (1∶400) to incubate overnight at 4 ℃, added the red Dylight 649 fluorescent labeled goat anti rabbit IgG secondary antibody (1∶400) to incubate for 1 h, added DAPI staining solution to incubate for 5 min, and observed the enrichment of complement around parasitophorous vacuoles under laser confocal microscopy. Take cobra venom factor (CVF) and inject it intraperitoneally into C57BL/6 mice as the CVF group; and set up a C3^-/- group (C3 whole gene knockout C3^-/- mice) and a control group (C57BL/6 mice). The mice in each group were challenged with 10 000 P. yoelii sporozoites. Total RNA was extracted from the liver and reverse transcribed to synthesize cDNA. The content of Plasmodium 18S rRNA was determined by fluorescence quantitative PCR, and the liver parasite burden were indicated as the relative content of 18S rRNA. Ten mice in control group (C57BL/6 mice) and C3aR^-/- group were challenged intravenously with 200 P. yoelii sporozoites, respectively, via tail vein. Five mice in control group (C57BL/6 mice), 6 C5aR whole gene knockout C5aR^-/- mice and 5 liver C5aR conditional knockout Alb-Cre^+/+C5aR^flox/flox hybrid mice were challenged with 1 000 P. yoelii sporozoites. Six mice in control group (C57BL/6 mice) and C5aR^-/- group were challenged with 1 000 P. berghei sporozoites. Tail vein blood was daily collected 3 d after challenge for making a thin blood smear and observed parasitemia after Giemsa staining, till all mice were founded malaria blood stages. Statistical analysis was conducted using Graphpad Prism 9.0 software. Normal distribution data is compared to continuous variables using t-tests, and pairwise differences are compared using one-way analysis of variance (ANOVA) for multiple comparisons; non normal distribution data are compared using Mann-Whitney U test. Results The PCR results showed that C3 (358 bp), C5 (267 bp) and their receptors C3aR (222 bp) and C5aR (388 bp) genes were amplified in Hepa1-6 cell line; C3 (202 bp), C5 (220 bp), C3aR (299 bp), and C5aR (374 bp) genes were amplified in HepG2-CD81 cell line. Western blotting results showed that both types expressed C3, C5, C3aR, and C5aR proteins. Laser confocal microscopy imaging showed that the cell nucleus showed blue fluorescence after DAPI staining, while the P. yoelii liver stages showed red spontaneous fluorescence. In the non permeable group, the highly expressed C3a and C5a in HepG2-CD81 cells show green fluorescence, overlapping with the distribution of parasitophorous vacuoles. The C5aR (pink) of the transmembrane group overlaps with the membrane of parasitophorous vacuoles (green); C5aR expression is still present on the membrane of parasitophorous vacuoles treated with C5aR antagonists. After infection with P. yoelii sporozoites, the relative content of 18S rRNA in the liver of the control group was 0.954 ± 0.523, the CVF group was 0.958 ± 0.231, and the C3^-/- group was 0.638 ± 0.437. There was no statistically significant difference among the three groups (P > 0.05). After infection with P. yoelii sporozoites, the control group and C3aR^-/- mice showed the blood stages after (5.30 ± 0.78) d and (5.30 ± 0.78) d, respectively. After infection with P. berghei sporozoites, the control group and C5aR^-/- mice showed the blood stages after (3.67 ± 0.47) d and (3.83 ± 0.69) d, respectively. There was no statistically significant difference in the appearance time of the the blood stages between the two groups of mice and the control group mice (P > 0.05). After infection with P. yoelii sporozoites, the appearance time of the blood stages in the control group, Alb-cre^+/+C5aR^flox/flox mice, and C5aR^-/- mice were (4.00 ± 0.00), (4.00 ± 0.00), and (4.17 ± 0.37) d, respectively, with no statistically significant difference (P > 0.05). Conclusion The activation of hepatocyte local complement had no significant effect on the development of Plasmodium liver-stage.

Analysis of laboratory detection capability for imported malaria in Hubei Province before and after malaria elimination

YI Jia, WU Dongni, DONG Xiaorong, ZHU Hong, LIN Wen, ZHANG Cong, SUN Lingcong

2024, 42(2):  177-181.  doi:10.12140/j.issn.1000-7423.2024.02.007

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Objective To understand the laboratory detection capability of imported malaria before and after malaria elimination in Hubei Province. Methods Data of malaria cases reported in Hubei Province from 2017 to 2022 were collected from National Infectious Disease Surveillance Reporting and Management System. The blood samples and detection review record forms of malaria cases reported in Hubei Province from 2017 to 2022 were collected. A review of the blood smears and blood samples was conducted by the professional technicians from the Hubei Provincial Malaria Diagnosis Reference Laboratory to identify malaria parasites. The blood smears were reviewed using microscopic examination, and the preparation, staining, and cleanliness of the blood smears were evaluated. The blood samples were reviewed for Plasmodium nucleic acids using fluorescence quantitative PCR. Using the retest results from the Provincial Malaria Diagnosis Reference Laboratory as the standard to analyze and compare the detection results provided by the malaria laboratories at the city (prefecture) levels on the positive qualification rate, parasite species coincidence rate, blood smear qualification rate, and sample delivery timeliness before (from 2017 to 2019) and after (from 2020 to 2022) malaria elimination. Results From 2017 to 2022, 501 cases of imported malaria were reported in Hubei Province, with 409 cases reported from 2017 to 2019 and 92 cases reported from 2020 to 2022. The sample collection rate from 2017 to 2019 was 99.3% (406/409), with a sample qualification rate of 99.3% (403/406). From 2020 to 2022, the sample collection rate was 94.6% (87/92), with a sample qualification rate of 82.8% (72/87). Among the 501 reported cases, 451 were confirmed as malaria by provincial malaria diagnosis review. From 2017 to 2019, a total of 373 confirmed malaria cases were reviewed, including 261 cases of falciparum malaria (70.0%), 48 cases of vivax malaria (12.9%), 52 cases of ovale malaria (13.9%), and 12 cases of malariae malaria (3.2%); from 2020 to 2022, a total of 78 confirmed malaria cases were reviewed, including 34 cases of falciparum malaria (43.6%), 20 cases of vivax malaria (25.6%), 19 cases of ovale malaria (24.4%), and 5 cases of malariae malaria (6.4%). There was a statistically significant difference in the composition of the 4 types of malaria before and after malaria elimination (χ² = 20.037, P < 0.05). The median sample submission time was 3 days (0-16 d) from 2017 to 2019, with an average of 4 days. From 2020 to 2022, the median sample submission time was 4 days (0-224 d), with an average of 8 days. The sample submission time was longer in all regions after malaria elimination (χ² = 25.591, P < 0.05). The positive compliance rates of the reported malaria cases in Hubei Province from 2017 to 2019 and from 2020 to 2022 were 91.9% (373/406) and 89.7% (78/87), respectively (χ² = 0.452, P > 0.05). The parasite specie compliance rates were 88.7% (331/373) and 78.2% (61/78), respectively, with a higher parasite compliance rate before malaria elimination (χ² = 6.296, P < 0.05). A total of 75 cases were misdiagnosed by city (prefecture) level malaria laboratories from 2017 to 2019, with a misdiagnosis rate of 18.5% (75/406), including 33 cases of qualitative misdiagnosis and 42 cases of species misdiagnosis; from 2020 to 2022, a total of 26 cases were misdiagnosed, with a misdiagnosis rate of 29.9% (26/87), including 9 cases of qualitative misdiagnosis and 17 cases of species misdiagnosis. There was a statistically significant difference in the species compliance rate for falciparum malaria before and after malaria elimination (χ² = 4.571, P < 0.05). A total of 492 blood smears were reviewed from 2017 to 2019, with a slide preparation qualification rate of 85.2% (419/492), staining qualification rate of 83.3% (410/492), and cleanliness qualification rate of 85.8% (422/492). From 2020 to 2022, a total of 209 blood smears were reviewed, with a slide preparation qualification rate of 71.8% (150/209), staining qualification rate of 76.1% (159/209), and cleanliness qualification rate of 76.6% (160/209). There were statistically significant differences in blood smear preparation, staining, and cleanliness evaluations before and after malaria elimination (χ² = 17.213, 5.054, 8.842, all P < 0.05). Conclusion After the elimination of malaria, the number of imported cases in Hubei Province has decreased. The laboratory detection capability of malaria has reduced slightly but remains stable.

Assessment of the quality of life of patients with echinococcosis in Gansu Province pre- and post-surgery based on the EQ-5D-5L questionnaire

LIU Zehang, ZHANG Xiaojuan, YANG Guobing, LEI Jiaxi, LIU Baixue, WANG Ying, WANG Zhiyi, WANG Dong, FENG Yu, WANG Liying

2024, 42(2):  182-190.  doi:10.12140/j.issn.1000-7423.2024.02.008

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Objective To analyse the changes in quality of life of patients with echinococcosis in Gansu Province pre- and post-surgery, and evaluate the impact of surgical treatments on the patients’ quality of life. Methods The patients with echinococcosis underwent surgery and were managed under the National Project for Echinococcosis Control and Prevention in Gansu Province were surveyed using questionnaire and EQ-5D-5L questionnaire to collect basic information, first visit record, surgical performance, and the quality of life pre- and post-surgery. The data were analyzed by SPSS 19.0 and Origin 2021. Results A total of 856 cases collected, including 417 males and 439 females. The age ranges from 7 to 85 years, with an average of 53 years. There were 737 cases of CE patients, 14 cases of AE patients, and 105 cases of unclassified patients. The longest time from the first surgery to postoperative follow-up for patients with echinococcosis was 17.5 years, and the shortest was 1 month. The average time interval between the investigation and the first surgery was 7.4 years (89 months). The preoperative and postoperative health utility values for patients with echinococcosis were 0.910 ± 0.142 and 0.967 ± 0.067, respectively, showing a 6.3% improvement postsurgery (Z = -12.081, P < 0.05). The preoperative and postoperative visual analog scale (VAS) scores were 79.10 ± 16.00 and 86.25 ± 11.64, respectively, showing a 9.0% improvement postsurgery (Z = -15.292, P < 0.05). The health utility value difference in patients with echinococcosis ranged between -0.308 and 1.348, the VAS score difference ranged between -55 and 90. A total of 42.8% (366/856) patients had an increase in health utility value postsurgery, and 62.7% (537/856) of patients had an increase in VAS score postsurgery. The proportion of patients with reduction of difficulties in the five dimensions was 19.1% (818/4 280), unchanged was 76.5% (3 275/4 280), and those with stable overall health had higher health utility values (Z = -16.580, P < 0.05) and VAS scores (Z = -8.606, P < 0.05). The proportion of patients with increased difficulties was 4.4% (187/4 280), the average age of first operation in the group with decreased health utility value was higher than that in another group (Z = 4.393, P < 0.05). The average age of first operation in the group with decreased VAS score was higher than that in another group (Z = 3.154, P < 0.05). The patients’ health utility value (F = 12.957, P_trend < 0.05) and VAS score (F = 10.808, P_trend < 0.05) showed a trend of positive linear correlation with the extension of postsurgery time. Conclusion Surgical treatment could impact the quality of life for patients with echinococcosis.

Effect of active ingredients of antiparasitic Chinese herb medicine on Echinococcus granulosus in vitro

LI Kunlei, XIA Jun, QIU Meiling, HU Meihe, JIGU Xiaoan, HOU Mengdan, ZHAI Shaohua

2024, 42(2):  191-198.  doi:10.12140/j.issn.1000-7423.2024.02.009

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Objective To evaluate the in vitro effects of water extracts and organic solvent crude extracts of antiparasitic Chinese herb medicine against Echinococcus granulosus protoscolices Methods The protoscolices were isolated from the liver cyst of sheep infected with E. granulosus and cultured for 24 hours in vitro to collect protoscolices with vitality greater than 95%, which were transferred into 96 well plate (100 μl with 100 protoscolices). In the culture wells, the water extracts of 11 antiparasitic Chinese herb medicines, including Brucea javanica, Melia toosendan, Polygonum hydropiper, Pericarpium granati, Quisqualis linn, Areca catechu, Cyrtomium fortunei, Derris trifoliata, Semen iridis, Galla chinensis and Agrimonia pilosa, were applied at final concentrations of 0.8 mg/ml (low concentration group), 1.6 mg/ml (medium concentration group), and 3.2 mg/ml (high concentration group), respectively. After 72 hours of treatment, the protoscolices vitality was detected by eosin staining, and morphology was observed under inverted microscope to calculate the mortality rate. In addition, an albendazole group with the same concentration and a blank control group were designated. The crude herb extracts from flavonoids, polysaccharides, saponins and alkaloids showing apparent anti-protoscolices effect were cultured with protoscolices at a final concentration of 1.00 mg/ml for 72 hours. The morphology of protoscolices was observed to calculate the mortality rate. At the same time, blank control group was set up. Liquid chromatography and tandem mass spectrometry analysis were performed for those crude extracts showing significant anti-protoscolices effects. Single variant ANOVA analysis was used for inter-group comparisons, and LSD-t-test was used for pairwise comparison. Results After 72 hours of treatment with the traditional Chinese medicine water extracts, the protoscolices edges appeared blurred, the structure was loose, and the matrix was dissolved in the high-concentration groups with B. javanica, M. toosendan and P. hydropiper water extracts. The high concentration group of the other 8 Chinese herb medicine water extracts showed normal protoscolices morphology and clear structures. The albendazole group showed mild damage to the germinal layer of the original scalp. The blank control group had normal protoscolices morphology. After 72 hours of in vitro culturing, the protoscolices mortality rates in B. javanica, M. toosendan and P. hydropiper water extracts high concentration groups were (99.63 ± 0.57)%, (90.89 ± 1.10)% and (51.93 ± 0.60)%; (85.97 ± 1.50)%, (81.14 ± 1.19)% and (42.46 ± 0.56)% in the medium concentration groups, and (78.34 ± 1.35)%, (77.27 ± 0.92)% and (36.66 ± 0.60)% in the low concentration groups, respectively. The difference was statistically significant compared with the blank control group [(0.62 ± 0.51)%] (F = 180 678.22, 41 488.99, 44 346.38; 19 543.86, 27 887.32, 34 590.79; 20 059.467, 41 953.68, 17 993.77; P < 0.01 for all comparations) and compared with the albendazole group [(30.03 ± 2.02)%] (F = 6 585.72, 4 210.84, 646.46; 2 956.80, 2 849.68, 210.83; 2 365.92, 2 712.28, 58.12; P < 0.01 for all comparetions). There were no statistically significant differences in the mortality rates of the protoscolices between the control group and the high-concentration groups, the medium-concentration groups and the low-concentration groups of the other 8 Chinese herb medicine water extracts (all P > 0.05). The protoscolices in the groups of B. javanica flavonoids, M. toosendan flavonoids, B. javanica saponins, P. hydropipe saponins and P. hydropiper alkaloid showed wrinkling, loose structure, and matrix dissolution， and the mortality rates of the protoscolices in the groups were (98.33 ± 2.89)%, (96.67 ± 5.77)%, 100%, (99.33 ± 1.15)% and （56.67 ± 2.11)%, respectively. The differences were statistically significant compared with the blank control group (10.33 ± 2.51)% (F = 1 584.00, 563.71, 3 808.47, 3 099.52, 14.65; P < 0.01 for all comparisons). There was no statistically significant difference in the mortality rate of the protoscolices between the high-concentration group of other crude extracts of traditional Chinese herb medicine and the control group (P > 0.05 for all comparisons). The liquid chromatography combined with tandem mass spectrometry analysis showed that 741 and 398 metabolites were identified in the positive and negative ion modes, respectively. Among them, four flavonoids with significant differences were identified in the flavonoids crude extracts of B. javanica in the positive ion mode, namely lutein, 5-methyl-7-methoxyisoflavone, catechins, and sweet orange flavonoids. Eight species were identified in negative ion mode, namely proanthocyanidin B2 and quercetin B3 β-D-glucoside, citrin, hesperetin, quercetin, luteolin, apigenin, and daidzein. Conclusion The water extracts of B. javanica, M. toosendan, P. multiflorum, as well as B. javanica flavonoids, M. toosendan flavonoids, B. javanica saponins and P. hydropipe saponins reveal significant anti-echinococcus granulosus protoscolices in vitro, and their effects are superior to those of albendazole.

Therapeutic effects of IL-1β receptor blocker on lung injury during the sensitization process of Echinococcus granulosus cyst fluid

WANG Chunsheng, WU Ergeli, XILIZATI Kulaixi, LI Yuqian, XIANYIDAN Abula, SUBI Tailaiti, PU Xueli, WANG Jialing, LI Meng, FANG Zhiyuan, YE Jianrong

2024, 42(2):  199-203.  doi:10.12140/j.issn.1000-7423.2024.02.010

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Objective To investigate the therapeutic mechanism of interleukin-1β (IL-1β) receptor blocker on lung injury in the sensitization process of Echinococcus granulosus cyst extravasated fluid. Methods The cysts of E. granulosus were collected from fresh sheep livers infected with E. granulosus from Hualing Slaughterhouse of Urumqi, Xinjiang. The cyst fluid was collected, digested and sedimented to obtain protoscoleces, and the supernatant was treated with endotoxin to obtain allergenic cyst fluid. Twenty-four mice were randomly divided into control group, sensitized group, blocker treatment group and blocker prevention group, with 6 mice in each group. Mice in the sensitized group, the blocker treatment group and the blocker prevention group were intraperitoneally injected with about 2 000 protoscoleces, respectively, while mice in the control group were injected with about the same volume (1 ml) of normal saline. Three months later, mice in the sensitized group were intraperitoneally injected with allergenic cyst fluid (0.1 ml per g weight); mice in the blocker treatment group were injected with allergenic cyst fluid prior to intravenous injection with IL-1β blocker (4 μg per g weight) 15 minutes later; mice in the blocker prevention group were injected with IL-1β blocker prior to intraperitoneal injection with allergenic cyst fluid 15 minutes later; mice in the control group were injected with the same volume of normal saline. The paraffin sections of mice lung tissues were stained with hematoxylin-eosin (HE) to observe the inflammatory damage under microscope. Total RNA was extracted from lung tissue and the mRNA relative transcription levels of tumor necrosis factor α (TNF-α), IL-6, cysteinyl aspartate specific proteinase-8, phosphatidylinositol 3 kinase (PI3K), protein kinase B (Akt) and nuclear factor kappa-B (NF-κB) were detected by qRT-PCR. Western blotting was used to detect the protein relative expression levels of PI3K, Akt and NF-κB, with β-actin as an internal reference. Data was analyzed by GraphPad Prism software. One-way analysis of variance was used for comparison between groups. Results HE staining showed that the lung tissues of the sensitized group were congested, with inflammatory cells swimming out and lymphocytes aggregating around the congested area. There was no obvious inflammatory reaction in the lung tissues of the blocker treatment group, blocker prevention group and control group. qRT-PCR results showed that the mRNA relative transcription levels of IL-6, TNF-α, caspase-8, PI3K, Akt and NF-κB were 1.057 ± 0.363, 1.020 ± 0.217, 1.004 ± 0.097, 1.000 ± 0.031, 1.035 ± 0.312 and 1.029 ± 0.304 in the control group, 2.013 ± 0.514, 2.189 ± 0.194, 6.433 ± 0.340, 1.594 ± 0.117, 2.902 ± 0.181 and 1.342 ± 0.146 in sensitized group, 1.243 ± 0.279, 1.268 ± 0.225, 0.869 ± 0.172, 1.103 ± 0.180, 1.371 ± 0.199 and 1.008 ± 0.202 in the blocker treatment group and 1.223 ± 0.358, 0.970 ± 0.303, 0.932 ± 0.298, 0.825 ± 0.404, 1.421 ± 0.137 and 1.083 ± 0.222 in the blocker prevention group, respectively. The mRNA relative transcription levels in the sensitized group were higher than the control group (t = 3.481, 2.759, 37.640, 2.237, 12.670, 2.274, all P < 0.05). The mRNA relative transcription levels in the blocker treatment group and blocker prevention group were lower than those in the sensitized group (t = 3.221, 7.593, 35.240, 5.610, 13.920, 3.287; 3.088, 8.299, 28.610, 4.475, 15.930, 2.390, all P < 0.05). Western blotting results showed that the protein relative expression levels of PI3K, Akt and NF-κB were 0.516 ± 0.075, 0.638 ± 0.103 and 0.198 ± 0.086 in the control group, 0.831 ± 0.061, 0.917 ± 0.069 and 0.784 ± 0.120 in sensitized group, 0.535 ± 0.108, 0.612 ± 0.206 and 0.247 ± 0.145 in the blocker treatment group and 0.526 ± 0.117, 0.565 ± 0.087 and 0.154 ± 0.031 in the blocker prevention group, respectively. The protein relative expression levels in the sensitized group were higher than the control group (t = 5.650, 3.901, 6.871, all P < 0.05). The protein relative expression levels in the blocker treatment group and blocker prevention group were lower than those in the sensitized group (t = 4.142, 2.434, 4.945; 4.013, 5.477, 8.821, all P < 0.05). Conclusion IL-1β receptor blocker may reduce the pulmonary inflammatory response through inhibiting PI3K/Akt/NF-κB pathway in the allergic reaction induced by E. granulosus cyst fluid, implying its therapeutic effects on lung injury.

Therapeutic effect of once and twice doses of albendazole for soil-transmitted helminth infection

YAN Xinliu, DU Zunwei, YANG Yaming, WANG Libo, WU Fangwei, LI Benfu, LI Hongbin, CHEN Ran, ZI Jinrong, PENG Jia, CAI Xuan, BAO Xueying, TANG Xiaoying, YANG Henglin

2024, 42(2):  204-210.  doi:10.12140/j.issn.1000-7423.2024.02.011

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Objective To evaluate the efficacy of once and twice doses of albendazole in treating human soil-transmitted helminth infections. Methods From October 2015 to January 2016, school students of grades 2-6 in 5 primary schools were selected to participate baseline survey (in Blang Mountain Township, Menghai County, Xishuangbanna Prefecture, Yunnan Province. Fecal samples were collected for two consecutive days (one sample per day), each of which was used to prepare three slides and examined using Kato-Katz method by microscopy. Students with fecal egg-positive finding (Ascaris, hookworm, Trichuris single or mixed infections) are treated with albendazole (400 mg single dose). Three days after treatment, fecal samples were collected in consecutive 2 days. After 4 weeks, the same group of students received additional single dose of 400 mg albendazole and their fecal samples were collected 3 days after dosing and examined. A questionnaire survey was conducted at the same time of two medications to assess the adverse reactions. Students who were still egg-positive after twice dosing were treated with albendazole once more. The examinees of the five primary schools were summarized according to administrative villages to evaluste the therapeutic effect on Trichuris infection in different villages. The comparison of rates is conducted using χ² test. Results A total of 438 people from 5 schools were investigated, and the infection rate of soil-derived nematodes was 82.19% (360/438). The infection rate was 79.19% (175/221) in boys and 85.25% (185/217) in girls, with no significant difference (χ² = 2.754, P > 0.05). The infection rates of soil-transmitted helminth were 89.79% (299/333), 90.57% (48/53), 21.43% (9/42), 4/10 (χ² = 133.781, P < 0.05) among the Blang, Lahu, Hani and other ethnological populations, respectively. The infection rates were 73.97% (54/73), 90.38% (94/104), 73.56% (64/87), 87.50% (28/32), 84.51% (120/142), respectively (χ² = 13.700, P < 0.05). The infection rates of Ascaris, hookworm and Trichuris were 52.51% (230/438), 35.39% (155/438) and 73.29% (321/438), respectively. The cure rates of Ascaris, hookworm and Trichuris infection after the first dose were 94.24% (180/191), 63.64% (77/121) and 2.64% (7/265), respectively. The cure rates of Ascaris, hookworm and Trichuris infection after the second dose were 98.77% (161/163), 91% (91/100) and 6.33% (14/221), respectively. There was a significant difference in the cure rates of patients with Ascaris, hookworm and Trichuris who took albendazole once and twice (χ² = 5.107, 22.487, 3.976, P < 0.05). There was no significant difference in the cure rates of mild, moderate and severe patients with Ascaris, hookworm and Trichuris after two doses (the first treatment χ² = 3.218, 3.930, 4.050, P > 0.05; the second treatment χ² = 1.745, 1.902, 0.329, P > 0.05). The incidence of adverse reactions such as difficulty swallowing pills, dizziness, nausea, diarrhea and vomiting after the first dose was 11.47% (39/340), 10.88% (37/340), 16.76% (57/340), 7.65% (26/340) and 1.47% (5/340), respectively. The incidence of adverse reactions in the second dose was close to that in the first dose, and there was no statistical significance in the incidence of adverse reactions between the two doses. According to the analysis of the village where the school was located, the cure rate of the students infected with Trichuris was 12.68% (9/71) in Kira Primary School after the second dose, which was higher than that in Mannian Primary School (3.60%, 4/111) (χ² = 5.374, P < 0.05). Conclusion Albendazole single therapy showed good effect on Ascaris infection, while double therapy presented a better deworming effect on Ascaris and hookworm infection without increasing the frequency of adverse reactions. It is unnecessary to increase the dosage of albendazole for treating moderate and severe Ascaris and hookworm infections. Albendazole is not effective for treating Trichuris, thus it is not advisable to use albendazole alone for treatment of Trichuris infection.

Study on the dynamic change of myeloid-derived suppressor cells proportion and cytokine expression in peripheral blood of mice infected with Echinococcus multilocularis

SU Yaxin, JIANG Nan, ZHANG Xiaocheng, WANG Ying, JIANG Xiaofeng, HUO Lele, WANG Yaxue, CAO Jianping, SHEN Yujuan

2024, 42(2):  211-216.  doi:10.12140/j.issn.1000-7423.2024.02.012

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Objective To analyze the dynamic changes in the proportion of myeloid-derived suppressor cells (MDSCs) and their subtypes in peripheral blood leukocytes of mice infected with Echinococcus multilocularis protoscoleces and detect the expression of cytokines related to MDSCs proliferation in the late-infected mice serum. Methods Twenty-four BALB/c mice were randomly divided into the infected group and the control group, with 12 mice in each group. Mice in the infected group were intraperitoneally injected with 1 200 E. multilocularis protoscoleces, while mice in the control group injected with the same volume of normal saline. Three mice from each group were randomly selected at 30, 90 and 180 days after infection to observe histomorphological changes in liver and spleen. Peripheral blood was collected from orbital venous plexus to prepare peripheral blood leukocytes. After incubation with externally labeled antibodies to CD11b, Gr-1, Ly6G and Ly6C, flow cytometry was used to detect the proportions of MDSCs, polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Serum samples from both groups were collected 180 days after infection for determine the concentrations of interleukin-6 (IL-6), IL-13, tumor necrosis factor α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) by ELISA assay. GraphPad Prism 9.0 software was utilized for graphical and statistical analysis; independent sample t-test was used for inter-group comparison. Results After E. multilocularis protoscoleces infection, cysts appeared in the liver of infected group mice 30 days after infection, and volume of cysts was increased over time. Spleen of infected group mice were enlaged over time 30, 90, and 180 days after infection. The results of flow cytometry showed that 30, 90 and 180 days after infection, the proportion of MDSCs in peripheral blood leukocytes of infected group were (13.2 ± 2.4)%, (15.7 ± 2.3)% and (41.3 ± 4.0)%, respectively. And the proportion of MDSCs in the control group were (12.4 ± 3.2)%, (6.0 ± 0.9)% and (22.3 ± 1.1)%, respectively. The infected group was significantly higher than the control group 90 and 180 days after infection (t = 3.949, 4.682; P < 0.05, 0.01). 30, 90 and 180 days after infection, the proportion of PMN-MDSCs in peripheral blood leukocytes of infected mice were (10.9 ± 2.1)%, (12.5 ± 2.4)% and (35.8 ± 3.6)%, respectively. And the proportion of PMN-MDSCs in the control group were (9.6 ± 3.1)%, (4.5 ± 0.6)% and (18.5 ± 0.6)%, respectively. The infected group was significantly higher than the control group 90 and 180 days after infection (t = 3.237, 4.788, P < 0.05, 0.01). 30, 90 and 180 days after infection, the proportion of M-MDSCs in peripheral blood leukocytes of infected mice were (1.8 ± 0.3)%, (1.1 ± 0.1)% and (4.6 ± 1.1)%, respectively. And the proportion of M-MDSCs in the control group were (2.3 ± 0.2)%, (0.5 ± 0.1)% and (3.4 ± 0.9)%, respectively. The infected group was significantly higher than the control group 90 days after infection (t = 3.246, P < 0.05). 180 days after infection, the concentrations of IL-6, IL-13, TNF-α and GM-CSF in the infected mice serum were (315.39 ± 13.58), (339.41 ± 13.35), (223.53 ± 27.49) and (262.31 ± 2.36) pg/ml, respectively, which were significantly higher than that of control group [(14.93 ± 0.55), (50.74 ± 0.88), (50.64 ± 1.64) and (115.28 ± 0.58) pg/ml] (t = 22.100, 21.580, 6.277, 60.460; P < 0.01, 0.01, 0.05, 0.01). Conclusion The proportion of MDSCs in peripheral blood leukocytes increased with time after E. multilocularis protoscoleces infection, mainly of PMN-MDSCs. The concentrations of serum IL-6, IL-13, TNF-α and GM-CSF increased of mice in the late stage of E. multilocularis protoscoleces infection, which may promote the proliferation and activation of MDSCs.

Transcriptome analysis of brain tissue in rat infected with Angiostrongylus cantonensis

CHENG Donghui, JIANG Tiange, JING Yidan, YANG Limin, GUO Yunhai, FANG Yuan, LI Zhongqiu, ZHANG Yi

2024, 42(2):  217-224.  doi:10.12140/j.issn.1000-7423.2024.02.013

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Objective To understand the expression levels in brain tissue transcriptome of rat infected with Angiostrongylus cantonensis. Methods Thirty-two SD rats were randomly divided into control group (12 rats) and infected group (20 rats). The rats in the infected group were infected with 40 A. cantonensis stage Ⅲ larvae by gavage and in the control group with the same volume of saline. 8 rats (3 of the control group and 5 of the infected group) were randomly dissected to collect brain tissues, of which paraffin sections were prepared and stained with hematoxylin-eosin (HE) to observe the pathological changes 1, 7, 14 and 21 days after infection. Brain tissue RNA was extracted 14 days after infection for detection of differentially expressed mRNA and lncRNA by using RNA sequencing technique. Gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway analysis of differentially expressed mRNA were performed. The STRING database was used to predict protein-protein interaction (PPI) between differentially expressed mRNA target proteins. Bioinformatics was utilized to construct the competitive endogenous RNA (ceRNA) regulatory network of differentially expressed genes. The differential expression of lncRNA was verified by qPCR. Results HE staining showed that pathological changes appeared in the rat brains of the infected group 14 days after infection with A. cantonensis, with cytoplasmic consolidation in the hippocampal neurons and parasite-like tissues could be seen at the meninges 21 days after infection. RNA sequencing result showed that the number of differentially expressed mRNAs in the rat brains was 955 (890 up-regulated and 65 down-regulated) 14 days after infection; the number of differentially expressed lncRNA was 193 (122 up-regulated and 71 down-regulated). GO enrichment analysis showed that differentially expressed mRNAs were mainly enriched in biological processes such as inflammatory response and immune response, the cellular components were mainly the extracellular space outside of the plasma membrane and the cell surface, and the molecular functions were mainly the chemokine activity and chemokine receptor binding. KEGG metabolic pathway analysis showed that the differentially expressed mRNAs were mainly involved in signaling pathways such as cytokine-cytokine receptor interactions and chemokines. PPI analysis showed that the main targets were chemokine ligand 11, RT1-Da, and serine family E member 1, all of which were associated with immune responses. ceRNA results showed that significantly enriched miRNAs such as mir-466b-3p, mir-1956, mir-207 and mir-328a-5p were associated with immune responses, apoptosis, angiogenesis and other processes. qPCR results showed that the H19 relative transcription level of rats in the infected group increased gradually, reaching a peak (15.074 ± 3.366) 21 days after infection, which was higher than rats in the control group (1.000 ± 0.113) (t = 13.190, P < 0.05). The relative transcript levels of RT1-CE6, LOC100910973 and lncR-ncf1 peaked 14 days after infection at 9.702 ± 1.408, 6.683 ± 1.299, and 7.733 ± 0.717, respectively, which were higher than rats in the control group (1.003 ± 0.039, 1.001 ± 0.156 and 0.999 ± 0.076) (t = 20.760, 13.830, 28.810, all P < 0.01). The AABR07030796.1 relative transcript level began to increase 14 days and peaked at 21 days after infection at 4.485 ± 0.236 and 5.068 ± 1.608, respectively, which were higher than rats in the control group (1.000 ± 0.159 and 1.001 ± 0.256) (t = 7.049, 8.229, both P < 0.01). The qPCR results and RNA sequencing results of H19, RT1-CE6, LOC100910973, lncR-ncf1 and AABR07030796.1 showed up-regulation in the rat brains of the infected group 14 days after infection with A. cantonensis with a consistent expression trend. Conclusion Total of 955 differentially expressed mRNA and 193 differentially expressed lncRNA were detected in the brain tissue of rats infected with A. cantonensis, which were mainly enriched in the inflammatory response, immune response and other biological processes.

Isolation, identification, and omics analysis of extracellular vesicles from Trichinella spiralis newborn larvae

LIU Yi, CAI Yuchun, CHEN Jiaxu, CHEN Shaohong, YU Yingfang

2024, 42(2):  225-233.  doi:10.12140/j.issn.1000-7423.2024.02.014

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Objective To isolate and identify the main components of extracellular vesicles (EV) secreted by Trichinella spiralis newborn larvae and elucidate the function of these EVs. Methods T. spiralis muscle larvae were sourced from the muscles of mice, which had been utilized for T. spiralis preservation. The small intestine was excised 5 days after infected with muscle larvae, and T. spiralis adults were obtained through mesh screening. These adults were then cultured in RPMI 1640 medium for 48 hours to yield newborn larvae. EVs were collected by differential ultracentrifugation from newborn larvae culture fluid of T. spiralis, and were observed morphologically under transmission electron microscope, and the diameter of the newborn larvae EVs was measured by nanoparticle tracking analysis. The EV protein components were verified using Western blotting, and analyzed by proteomic mass spectrometry. RNA sequencing technology was applied to analyze the miRNA sequences of the newborn larvae and their secreted EVs. The differences of protein components and differential expression between newborn larvae and their EVs were compared using t-test. The differentially expressed proteins and miRNA target genes were analyzed by gene ontology (GO) functional item enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. WoLFPSORT was used to analyze the subcellular localization of proteins. Results Transmission electron microscopy and nanoparticle tracking analysis revealed that T. spiralis newborn larvae produce cup-shaped, membranous vesicles with a diameter of approximately (101.7 ± 1.8) nm. Western blotting demonstrated that the newborn larvae-derived EVs generate specific bands when reacted with sera from mice infected with Trichinella spiralis, with a approximate molecular weight of 65 000. Through proteomics mass spectrometry, 1 424 and 231 proteins were detected in the newborn larvae and EV samples, respectively. Among these, 220 proteins were expressed in both groups, with 116 displaying differential expression patterns (31 upregulated and 85 downregulated in EVs). The subcellular localization analysis revealed that the differentially expressed proteins were predominantly localized in the cytoplasm (30.2%, 35/116), nucleus (19.0%, 22/116), and cell membrane (17.2%, 20/116). miRNA sequencing data showed that 183 and 117 miRNAs were detected in newborn larvae and EV samples, respectively; 46 miRNAs were found in both groups, with 40 showing differential expression patterns (10 upregulated and 30 downregulated in EVs). Notably, let-7-5p, miR-125-5p, miR-10, and miR-92 exhibited higher levels of upregulation in newborn larvae-derived EVs. The GO functional enrichment analysis indicated that the differentially expressed proteins could be categorized into three main groups: cell composition (57), molecular function (73), and biological processes (63). The target genes of differentially expressed miRNA were primarily associated with autophagy-related genes such as transferase activation of autophagy-related 12 and ubiquitin linkage enzyme complex. KEGG pathway enrichment analysis suggested that the differentially expressed proteins were significantly enriched in metabolic pathways and protein processing in endoplasmic reticulum-related pathways, whereas the target genes of differentially expressed miRNA were only involved in the autophagy-other pathway. Conclusion This study successfully isolated and identified the EVs secreted by newborn larvae of T. spiralis. 116 differentially expressed proteins and 40 miRNAs were identified between the newborn larvae and their EVs, predominantly participating in metabolic, autophagic and other pathways, which inferring that newborn larvae EVs may play important roles in evading host immune defence at early Trichinella infection.

Establishment of a rapid detection method of mosquito-borne dengue virus based on loop-mediated isothermal amplification microfluidic chip technology

JIANG Ning, BAI Jie, LI Ping, SHAN Wenqi, ZHOU Qiuming, DONG Haowei, YUAN Hao, TAO Feng, LI Xiangyu, MA Yajun, PENG Heng

2024, 42(2):  234-241.  doi:10.12140/j.issn.1000-7423.2024.02.015

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Objective To establish a loop-mediated isothermal amplification (LAMP) based microfluidic chip technology for the rapid detection of dengue virus in mosquitoes. Methods According to the gene fragments encoding dengue virus capsid protein (C), pro-membrane protein (prM) and non-structural protein 2A (NS2A), plasmids pUC-GW-Amp-CprM and pUC-GW-Amp-NS2A were constructed, and specific primer sets for multi-group loop-mediated isothermal amplification were designed. The LAMP microfluidic chip was prepared for amplification with the target gene plasmids and dengue virus nucleic acid used as templates. Optimal primer combinations were screened according to the peak time, fluorescence intensity, false positivity, reproducibility, and sensitivity of the assay. The optimal primer combinations were used to detect the cDNAs of major dengue vectors, including Aedes aegypti and A. albopictus and commonly seen Culex quinquefasciatus, Anopheles sinensis, and Chironomus. The combinations were also used for cross-examination with common mosquito-borne pathogens, such as Zika virus, epidemic B encephalitis virus, Plasmodium falciparum, and Yellow fever virus to evaluate the specificity of the LAMP assay. The LAMP assay sensitivity for detection of pUC-GW-Amp-CprM and pUC-GW-Amp-NS2A plasmids was evaluated by comparing with the results of PCR and qPCR, respectively. Dengue virus RNA was mixed with their vector A. aegypti and A. albopictus RNA at 1 ∶ 10, 1 ∶ 100, and 1 ∶ 1 000, respectively, to simulate samples of vector carrying dengue viruses for LAMP microfluidic microarray detection. The A. albopictus cDNA samples collected from five locations, including Sandu Island in Ningde City, Xi’an City, Zhujiajiao, Changxing Island and Wujiaochang in Shanghai, were examined. Results The primer screening results suggested that the CprMG2 and NS2AG1 primers were identified as the optimal primer combinations because of their early peak onset, high relative fluorescence unit and highest sensitivity. Among them, CprMG2 can detect pUC-GW-Amp-CprM plasmid templates at concentrations as low as 10^-6 ng/μl, with a minimum detection limit of 1.3 × 10³ copies, and NS2AG1 can detect pUC-GW-Amp-NS2A plasmid templates at concentrations as low as 10^-9 ng/μl, with a minimum detection limit of 1 copy. The optimal primer combinations CprMG2 and NS2AG1 showed no unspecifical amplification of arthropod cDNAs of A. aegypti and A. albopictus and other common arthropods, including C. quinquefasciatus, An. sinensis, and Chironomus. They did not cross-react with the nucleic acids of the Zika virus, encephalitis B virus, P. falciparum and Yellow fever virus. The lowest detection limit of CprM target gene detected by LAMP microfluidic chip was 1.3 × 10³ copies, which was ten folds more sensitive than that of PCR (1.3 × 10⁴ copies); the lowest detection limits of CprM and NS2A target genes detected by LAMP microfluidic chip were 1.3 × 10³ and 1 copies, which were equivalent to those of qPCR (1.3 × 10³ and 1 copies). The LAMP microfluidic chip testing results on the simulated field samples showed that CprMG2 could detect 1 ∶ 10, 1 ∶ 100 and 1 ∶ 1 000 dilutions of the virus, and NS2AG1 could detect a minimum of 1 ∶ 100 dilutions of the virus, with a specificity of 100%. LAMP microfluidic chip testing of A. albopictus collected in the five field sites yielded negative results. Conclusion A LAMP microfluidic chip method for dengue virus detection was established. The method shows high sensitivity and specificity, short detection time, and can be used for field detection of mosquito vectors cattying dengue virus.

Infection of Blastocystis and Cyclospora cayetanensis in residents in Eryuan County, Yunnan Province and their molecular characterization

WANG Yaxue, JIAN Jinhua, LIU Hua, QIN Yuan, PENG Xiaoxue, SU Yaxin, SHEN Yujuan

2024, 42(2):  242-250.  doi:10.12140/j.issn.1000-7423.2024.02.016

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Objective To investigate the prevalence and molecular characterizations of Blastocystis and Cyclospora cayetanensis in residents in Eryuan County, Yunnan Province to provide reference for parasitic disease control and prevention. Methods From July to August 2022, permanent residents over the age of 1 were enrolled for the survey by simple random sampling from the five villages in Eryuan County, Yunnan Province. Fresh fecal samples and their social demographic information were collected. The fecal genomic DNA was extracted, and the ribosomal small subunit RNA (SSU rRNA) genes of Blastocystis and C. cayetanensis were amplified by PCR and nested PCR, respectively, in order to analyze the prevalence in the residents. The positive products were sequenced bidirectionally, the species and gene subtypes were identified by BLAST alignment in NCBI. The phylogenetic trees were constructed using the neighbor-Joining method by MEGA 11.0 software. SPSS 21.0 software was used for statistical analysis, and the infection rates were compared by χ² test or Fisher’s exact probability method. Results A total of 100 fecal samples were obtained from residents, of which the Blastocystis infection rate was 14.0% (14/100). Among them, the Blastocystis infection rates in male and female were 14.6% (6/41) and 13.6% (8/59), respectively. Residents over 60 years old and the family size of 6 persons and above had the highest infection rate, which was 18.5% (5/27) and 3/15, respectively. The infection rates were 2/11 and 17.0% (8/47) for those with no education and only elementary school education, respectively. All the infected residents were farmers. There was no statistically significant difference in Blastocystis infection rate by gender, age, family size, education level and occupation (χ² = 0.023, 2.730, 2.235, 1.404, 1.668; all P > 0.05). The C. cayetanensis infection rate was 2.0% (2/100). All of the infected residents were female farmers. No mixed infection of Blastocystis and C. cayetanensis was detected. Both 7 ST1 and ST3 sequences were amplified from 14 Blastocystis-positive samples, of which 7 ST1 sequences had 25 nucleotide differences. Five sequences were 100% homology to the Blastocystis sequences (GenBank: ON932511, KU147348, MK801408, MW728079, OR754904); 2 were new sequences (GenBank: PP439288-PP439289), with 99.8% homology to the Blastocystis sequences (GenBank: OP725964, KU147333). There was 1 nucleotide difference within 7 ST3 sequences, with 100% homology to the Blastocystis sequences (GenBank: KU147372, MK801366). The 2 sequences of C. cayetanensis obtained were identical and showed 100% homology to the C. cayetanensis sequence (GenBank: KY770755). The phylogenetic trees showed that Blastocystis in this study clustered in large branches with gene subtype ST1 and ST3, respectively, and C. cayetanensis clustered in the same branch with C. cayetanensis of human origin. Conclusion Blastocystis and C. cayetanensis were detected in the residents of Eryuan County, Yunnan Province. The infection rate of Blastocystis was high, with the subtypes of ST1 and ST3, and the ST1 of Blastocystis reveals higher genetic variation.

Molluscicidal efficacy of gallic acid in combination with niclosamide

ZHENG Tao, LIU Jiahao, LI Bin, LI Jiashan, NIE Juan, XIONG Tao

2024, 42(2):  251-258.  doi:10.12140/j.issn.1000-7423.2024.02.017

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Objective To investigate the combined snail killing effect and mechanism of traditional Chinese medicine monomers gallic acid and niclosamide, which have alternating oxidase (AOX) inhibitory activity. Methods Negative snails were collected from Gong’an County, Hubei Province and randomly divided into 7 groups: a blank control group (H₂O), experimental groups were treated with niclosamide (N1 group: 0.06 mg/L, N2 group: 0.1 mg/L) and gallic acid (G1 group: 0.8 g/L, G2 group: 1.6 g/L) alone or in combination (M1 group: 0.06 mg/L niclosamide + 0.8 g/L gallic acid, M2 group: 0.06 mg/L niclosamide + 1.6 g/L gallic acid). Detect the survival rates of each group of snails after drug immersion for 12, 24, and 48 hours. Slice and stain them after liquid nitrogen embedding, observe and quantitatively analyze them under an optical microscope. Measure the relative enzyme activity values of cytochrome C oxidase (CCO) and lactate dehydrogenase (LDH) in the soft sections of snails, expressed as average optical density values. After homogenizing the snail, centrifuge and take the supernatant. Incubate with DCFH-DA probe, determine the total protein content using a BCA protein quantification kit, detect fluorescence intensity (FI) using a multifunctional enzyme-linked immunosorbent assay (ELISA), and divide the ROS value by the FI measured in the sample by the protein concentration (μg). The value represents the determination of superoxide dismutase (SOD) levels using a total SOD activity detection kit. The difference analysis of snail mortality rate was conducted using χ² test. After determining the homogeneity of variance using Levene’s Test, the data on CCO activity and oxidative stress levels were compared pairwise between different groups using Tukey HSD’s multiple comparison method. Results The G1 and G2 groups did not show significant snail killing effects after immersion. The mortality rates of snails in the M1 and M2 groups after 48 h of immersion reached 70.0% (56/80) and 84.2% (101/120), respectively, higher than those in the N1 group (44.4%, 32/72) (χ² = 9.13, 32.52; P < 0.05); Compared with the N2 group, there was no statistically significant difference in snail mortality rate (χ² = 0.11, 2.58; P > 0.05). The LDH activity of snails in N1 and N2 groups showed a decreasing trend; The activities of CCO and LDH in the screw bodies of M1 and M2 groups decreased (P < 0.05). After 48 hours, the LDH activities in muscle tissue and liver of M1 group were 0.152 ± 0.002 and 0.172 ± 0.016, respectively, and the CCO activities were 0.180 ± 0.022 and 0.335 ± 0.014, respectively; The LDH activity of the M2 group was 0.166 ± 0.008 and 0.173 ± 0.022, and the CCO activity was 0.199 ± 0.009 and 0.294 ± 0.015, respectively. There was a statistically significant difference (P < 0.05) between the two groups and the blank control group (LDH activity was 0.229 ± 0.006 and 0.227 ± 0.010, and CCO activity was 0.259 ± 0.008 and 0.428 ± 0.024, respectively). The SOD activity of the M1 group at 24 hours after treatment was (5.56 ± 0.91) UI/g, which was higher than that of the blank control group at (5.26 ± 0.08) UI/g (P < 0.05); The SOD activity of the M2 group showed a trend of first increasing and then decreasing [(2.40 ± 0.45), (8.14 ± 0.15), (1.60 ± 0.21) UI/g] at 12, 24 and 48 h after treatment. Compared with the UI/g of the blank control group at the corresponding time points [(3.54 ± 0.94), (5.26 ± 0.08), (5.10 ± 0.87) UI/g], the trend of change was significant (P < 0.05). The ROS levels in the M1 group were (1 619.00 ± 168.25) FI/μg and (1 866.65 ± 211.79) FI/μg, respectively, at 24 and 48 h after treatment. The ROS levels in the M2 group reached (2 451.29 ± 195.91) FI/μg after 48 hours, which were higher than those in the blank control group at the corresponding time points [(802.37 ± 114.69), (1 393.81 ± 86.12) FI/μg] (P < 0.05). Conclusion Gallic acid significantly enhances the snail killing effect of low concentration chloramphenicol. By blocking the compensatory upregulation of AOX, it further exacerbates the energy metabolism imbalance and oxidative stress in snails. This may be an important mechanism by which gallic acid has an auxiliary snail killing effect.

Research on the progress of programmed cell death in echinococcosis

DAWA Zhuoma, LIU Chuanchuan, FAN Haining

2024, 42(2):  259-266.  doi:10.12140/j.issn.1000-7423.2024.02.018

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Echinococcosis is a serious zoonosis disease caused by the metacestode stage larval of Echinococcus granulosus and E. mulculoris. Humans as an intermediate host infected by the larvae stages of the parasites through accidental ingestion of the eggs. Programmed cell death is common in the growth and development of Echinococcus. Programmed cell death includes apoptosis, pyroptpsis, autophagy and ferroptosis. In this review, we summarized the role of programmed cell death pathways in the echinococcosis and try to provide a basic theoryto explore potential targets for the prevention and treatment of echinococcosis.

SHORT COMMUNICATION

The analysis of the epidemiological characteristics and the diagnosis of imported malaria before and after the COVID-19 pandemic in Nanjing City

HE Yisha, XIE Chaoyong, WANG Yu, LI Yanjing

2024, 42(2):  267-271.  doi:10.12140/j.issn.1000-7423.2024.02.019

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To analyze the epidemiological characteristics and diagnosis of imported malaria before and after the COVID-19 pandemic in Nanjing City, data pertaining to the epidemic situation and case investigation of malaria in Nanjing City from 2017 to 2022 were collected from the National Notifiable Communicable Disease Reporting System to analyze the source of infection, distribution and diagnosis and treatment of malaria reported cases. A total of 89 malaria cases were reported in Nanjing City from 2017 to 2022, and all were imported cases. Among them, falciparum malaria accounted for 79.8% (71/89). A total of 71 and 18 malaria cases were reported before and after COVID-19 epidemic, respectively, and falciparum malaria accounted for the highest proportion before (78.9%, 56/71) and after the epidemic (15/18). The reported cases mainly acquired Plasmodium infections in African regions， with accounted for 94.4% (67/71) before and 18/18 after the epidemic. The reported cases were distributed in each month from 2017 to 2019, and the number of reported cases peaked in January (12 cases). There were no significant seasonal variations in the distribution of reported malaria cases from 2020 to 2022. The number of reported cases peaked in November (5 cases). The reported cases were mainly young and middle-aged males and labour workers before and after the COVID-19 pandemic. The proportion of international students was 15.5% (11/71) before the pandemic, and decreased to 0 after the pandemic (χ² = 5.352, P < 0.05). From 2017 to 2019, the first treatment hospitals and diagnosis hospitals were mainly city-level hospitals, accounting for 62.0% (44/71) and 77.5% (55/71), respectively. From 2020 to 2022, the hospitals for initial treatment and final diagnosis were mainly county-level hospitals, accounting for 15/18 and 12/18, respectively. The proportion of confirmed cases in county-level hospitals was 8.4% (6/71) before the COVID-19 pandemic and increased to 12/18 after the epidemic. The proportion of malaria cases with definitive diagnoses upon admission increased from 42.2% (30/71) before the COVID-19 pandemic to 13/18 after the pandemic (χ² = 26.663, 5.165, all P < 0.05). From 2017 to 2019, the average and median time from the onset of symptoms to the initial treatment for each case were 2.8 d and 2.0 d, respectively. The average and median time from the initial visit to diagnosis were 1.3 d and 1.0 d, respectively. From 2020 to 2022, the average and median time from the onset of symptoms to the initial clinical visit were 1.5 d and 1.0 d, respectively. The average and median time from initial clinical visit to diagnosis were 0.3 d and 0 d, respectively. The interval between the onset of symptoms to initial clinical visit and the time of initial clinical visit to final diagnosis before the pandemic was significantly longer than that after the epidemic (Z = -2.359, -2.658, all P < 0.05). Higher efficiencies for the initial treatment and diagnosis of the malaria cases were observed. The diagnosis of malaria cases in county-level hospitals has been improved after the COVID-19 pandemic in Nanjing City.

CASE REPORTS

Diagnosis of a case of Giardia lamblia infection

MIN Xiangyang, FENG Meng, ZHAO Xuhong, WENG Wenhao, LI Hanhua

2024, 42(2):  272-274.  doi:10.12140/j.issn.1000-7423.2024.02.020

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A 40-year-old male, who is a Shanghai resident, presented to the emergency department of Yangpu Hospital, School of Medicine, Tongji University in July 2018. The patient complained of watery diarrhea for 1 day. The patient had a travel history to Panama one month before the onset of the disease and had no history of drinking unboiled water or consuming uncooked meat. The routine blood cell count results were normal except for an increase in C-reactive protein of 16.17 mg/L (normal value 0-10 mg/L), which indicated an infection. Giardia trophozoites and cysts were found in stool specimens by microscopic examination. The IFA test results showed that G. lamblia trophozoites was recognized by the patient’s serum (1 ∶ 50), and there was strong fluorescence staining on the surface of G. lamblia. A 147 bp fragment of Giardia-specific 18S ribosomal RNA was amplified by PCR. The sequence of this fragment was 100% consistent with the sequence of G. lamblia assemblage A (GenBank login number: KY706490), and it was clustered on the same branch with G. lamblia assembly A in the phylogenetic tree constructed by the neighbour-joining method. Based on the patient’s clinical manifestations and relevant examination results, a G. lamblia infection diagnosis was made. The patient was treated with oral metronidazole at 20 mg/kg daily, divided into 3 doses and take for 7 days. Blood and fecal routine tests were re-examined two weeks later, and both results returned to normal. The patient’s condition was improved without recurrence of symptoms.

A human Babesia infection case characterized by multiple organ infarctions

XU Hui, FEI Yan, GONG Zhihong, XIE Shuying, XU Yun, LIU Junpu, XIE Jingzi, ZHOU Feng, XIE Huiqun

2024, 42(2):  275-278.  doi:10.12140/j.issn.1000-7423.2024.02.021

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The 58-year-old female patient was a farmer from Yuanzhou District, Yichun City, Jiangxi Province. The patient underwent radical pancreaticoduodenectomy for cholangiocarcinoma in the Second People’s Hospital of Yichun City on June 4, 2023 due to postoperative intra-abdominal hemorrhage. After blood transfusion and other treatments, the active hemorrhage remained. She underwent cesarean section on June 9, with recurrent abdominal pains after the operation. She had symptoms of vomiting and fever with the highest temperature of 40 ℃ in the middle and late July. The patient was transferred to the Second Affiliated Hospital of Nanchang University with “abdominal infection” diagnosis on August 13. Laboratory tests showed the red blood cell count was 1.75 × 10¹²/L and the hemoglobin was 49 g/L, which suggested severe anemia. The total protein was 60.5 g/L and the albumin was 31.4 g/L, which suggested hypoproteinemia. The patient also had mildly elevated total, direct and indirect bilirubin. Abdominal CT examination showed splenomegaly, splenic multiple infarct foci. Cranial diffusion imaging examination showed multiple infarct foci in the center of the bilateral semiovals and the lateral paraventricular region. Giemsa stained blood smear on August 15 showed ring-form parasites in some red blood cells, and parasitic infections were considered. The patient has been living in the rural area of Yuanzhou District for a long time without domestic or overseas-traveling history, and no clear history of tick bites before the onset of the disease. The DNA extracted from blood samples of August 17 and 18 was amplificated uesd Babesia spp. specific primer by PCR and sequenced to identify 18S rRNA gene fragment. The PCR product identified with 99.57% homology to B. microti (GenBank: JX962781.1). The diagnosis of Babesia microti infection was confirmed. The patient was treated with oral clindamycin 0.45 g, 3 times per day for 5 days, and the symptoms were relieved. On September 30, parasites were still detected on microscopic examination. The patient was treated with doxycycline hydrochloride injection 0.1 g per day for 3 days, followed by doxycycline hydrochloride tablets 0.1 g, 2 times per day for 15 days. At follow-up on October 30, the patient had only a slight tremor in the upper limb.