Current Issue
30 October 2022
Volume 40 Issue 5
EXPERT VIEWPOINT
The toxicological and pharmacological effects of parasite-derived components on the host
XU Zhi-peng, JI Min-jun, WU Guan-ling
2022, 40(5):  561-571.  doi:10.12140/j.issn.1000-7423.2022.05.001
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In recent years, a new translational medicine proposition, the toxicological and pharmacological effects of parasite-derived components on the host, has received extensive attention. Therefore, an in-depth understanding of parasite-derived molecules’ toxicological and pharmacological effects from the perspective of infection immunology can deepen our understanding of the pathogenesis of parasitic diseases but also facilitate research and development based on the concept of translational medicine. The transformational application value of parasite-derived molecules has become and will continue to be one of the most fantastic research tasks in the field of parasite research. This review focuses on recent advances in the pharmacological effects of zoonotic parasites and their derived components in inducing and regulating host immunometabolism-related diseases, and explores their potential mechanisms.

Integrated surveillance and early warning system for emerging infectious diseases based on One Health concept: structures and innovations
LI Hui-min, LIU Jing-shu, WANG Xi-han, ZHAO Han-qing, XIE Yi, YIN Jing-xian, LV Chao, ZHOU Nan, JIANG Tian-ge, GU Si-yu, YIN Kun, ZHOU Xiao-nong, GUO Xiao-kui, HU Qin-qin
2022, 40(5):  572-578.  doi:10.12140/j.issn.1000-7423.2022.05.002
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One Health is an upgrade and optimization of health concepts, which recognizes the integrated health of the human-animal-environment. It emphasizes the use of interdisciplinary collaboration, multi-sectoral coordination, and multi-organizational One Health approaches to solve scientific questions. The surveillance and early warning system is the basis of public health emergency prevention and control. The COVID-19 pandemic and the emerging infectious disease (EID) have put great challenges on the existing surveillance and early warning systems worldwide. Guided by the concept of One Health, we attempt to build a new pattern of integrated surveillance and early warning system for EID. We will detail the system including the One Health-based organizational structure, zoonotic and environmental science surveillance network, EID reporting process, and support and guarantee from education and policy. The integrated surveillance and early warning system for EID constructed here has practical and application prospects, and will provide guidance for the prevention and control of COVID-19 and the possible EID in the future.

ORIGINAL ARTICLES
Effects of ROP16 protein of Toxoplasma gondii on polarization and apoptosis of MH-S cells and their related mechanisms
LI Jia-ming, WANG Yi-xuan, YANG Ning-ai, MA Hui-hui, LAN Min, LIU Chun-lan, ZHAO Zhi-jun
2022, 40(5):  579-586.  doi:10.12140/j.issn.1000-7423.2022.05.003
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Objective To investigate the expression of Toxoplasma gondii rhoptry protein 16 (ROP16) protein in mouse alveolar macrophages (MH-S) and its affect on cell polarization and apoptosis and the mechanisms involved. Methods MH-S cells transfected with ROP16 overexpression lentivirus labeled of green fluorescent were used to construct ROP16-MH-S cell line capable of stable expression of ROP16 (overexpression group). A blank vector lentivirus transfection control group (blank vector group) and no transfection control group (control group) were assigned in parallel. The expression of ROP16 in ROP16-MH-S and its localization within the cells were detected by immunofluorescence 72 h post-transfection. With the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal reference, and the mRNA relative transcription level of pro-inflammatory (M1) factor interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-18, IL-6 and IL-12; apoptosis-suppressing gene B-cell lymphoma/leukemia-2 (Bcl-2); and anti-inflammatory (M2) factors IL-10, transforming growth factor (TGF)-β, pro-apoptotic genes Bcl-2-associated X protein (Bax), cysteine protease 3 (Caspase 3), Caspase 9 in ROP16-MH-S cells was detected by RT-qPCR. The relative expression level of polarized protein arginase 1 (Arg-1), signal transducer and activator of transcription 3 (STAT3), phosphorylation at site 705 (pTyr705)-STAT3, STAT6, pTyr641-STAT6 and apoptosis proteins Caspase 9, Caspase 3, Bcl-2 and Bax in ROP16-MH-S cells were detected by Western-blotting. The apoptosis of ROP16-MH-S cells was detected by flow cytometry. One-way analysis of variance was used for comparison between groups. Results Strong green fluorescence was seen in the blank vector group and overexpression group at 72 h after transfection. Significant red fluorescence was also observed in the nucleus of ROP16-MH-S cells and its surrounding in the overexpression group. RT-qPCR results showed that the relative transcription level of ROP16 mRNA in the overexpression group was 8 023.459 ± 39.325 with green fluorescence, which was higher than that in the blank vector group (5.540 ± 0.001) (F = 83 188, P < 0.01); Western-blotting showed that the relative expression level of ROP16 protein in the overexpression group was 16.349 ± 0.746, which was higher than that in the blank vector group (1.291 ± 0.333) (F = 831.7, P < 0.01). RT-qPCR results showed that the relative transcription levels of pro-inflammatory (M1) factors IL-1β, TNF-α, IL-18, IL-6 and IL-12 in the overexpression group were 0.495 ± 0.002, 0.337 ± 0.007, and 0.378 ± 0.014, 0.474 ± 0.035, and 0.730 ± 0.021, respectively, which were lower than those in the blank vector group (0.994 ± 0.043, 1.165 ± 0.034, 0.943 ± 0.005, 1.153 ± 0.028, 0.926 ± 0.031) (F = 261.7, 536.5, 1 682.0, 225.0, 78.5; P < 0.01); the relative transcription levels of anti-inflammatory (M2) factors IL-10 and TGF-β mRNA were 7.013 ± 0.032 and 1.608 ± 0.024, respectively, which were significantly higher than those in the blank vector group (0.790 ± 0.031, 1.091 ± 0.027) (F = 23 835.0, 200.1, P < 0.01). Western-blotting revealed that that the relative expression levels of Arg-1, pTyr705-STAT3, and pTyr641-STAT6 proteins in the overexpression group were 2.337 ± 0.089, 3.471 ± 0.046, 3.905 ± 0.045, respectively, which were higher than those in the blank vector group (0.871 ± 0.014, 1.482 ± 0.071, 1.514 ± 0.050) (F = 640.8, 1 608.0, 3 528.0, P < 0.01). The flow cytometry results showed that the apoptosis rate in the overexpression group was (2.990 ± 0.042)%, which was lower than that in the control group (6.480 ± 0.071)% and the blank vector group (5.655 ± 0.290)%, respectively (F = 219.7, P < 0.01). Western-blotting showed that the relative expression levels of the pro-apoptotic proteins Bax, Caspase 3, Caspase 9 and Bax/Bcl-2 in the cells of the overexpression group were 0.558 ± 0.005, 0.640 ± 0.011, 0.593 ± 0.026 and 0.453 ± 0.011, respectively, which were lower than those in the blank vector group (0.991 ± 0.010, 0.926 ± 0.006, 0.963 ± 0.012, 0.834 ± 0.008) (F = 2 850.0, 1 200.0, 359.3, 2 337.0, P < 0.01). RT-qPCR showed that the relative transcription levels of pro-apoptotic genes Bax, Caspase 3, Caspase 9 and Bax/Bcl-2 mRNA in the overexpression group were 0.588 ± 0.086, 0.563 ± 0.025, 0.403 ± 0.014 and 0.158 ± 0.008, respectively, which were lower than those in the blank vector group (0.924 ± 0.016, 0.937 ± 0.041, 0.807 ± 0.032, 0.779 ± 0.014) (F = 24.7, 78.6, 154.9, 265.5, P < 0.01); while the relative transcription level of restraining-apoptosis gene Bcl-2 was 3.702 ± 0.362, which was higher than that in the blank vector group (1.186 ± 0.006) (F = 104.1, P < 0.01). Conclusion T. gondii ROP16 protein is stably expressed in ROP16-MH-S cells, mainly located in and around the nucleus,activating STAT3 and STAT6, phosphorylate Tyr705-STAT3 and Tyr641-STAT6, regulating ROP16-MH-S cells towards M2 polarization and thereby suppressing cell apoptosis.

Preparation and application of monoclonal antibody against Toxoplasma gondii bradyzoite antigen 1
ZOU Wei-hao, WU Wei-ling, LIAO Yuan-peng, CHEN Min, PENG Hong-juan
2022, 40(5):  587-593.  doi:10.12140/j.issn.1000-7423.2022.05.004
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Objective To prepare monoclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (BAG1) and explore its application in identification of T. gondii bradyzoite. Methods The bradyzoites of T. gondii ME49 strain (in vitro induced) after induction in alkaline medium (pH 8.2) for 5 days and the ME49 strain obtained from mouse brain tissue homogenate (in vivo formed) 2 months after chronic infection were collected. Total RNA was extracted from the in vitro induced T. gondii ME49 bradyzoite and amplified by RT-PCR for the bag1 open reading frame segment, which was subcloned into the prokaryotic expression vector pET-28a(+) and transformed into BL21 (DE3) Escherichia coli. The recombinant E. coli was induced by 1 mmol/L IPTG to express BAG1 protein, which was purified by Ni-NTA column, and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Six BALB/c mice were immunized subcutaneously with 20 μg purified BAG1 protein three times (Freunds complete adjuvant was used for the primary immunization, and the same amount of Freunds incomplete adjuvant was used for two boosting at an interval of two weeks). The mice with a high antibody titer were selected to collect spleen tissues for separating splenocytes, which were fused with SP2/0 myelomacells. The positive hybridoma cells were screened by indirect ELISA using purified BAG1 protein, and then underwent multiple subcloning screening for the hybridoma cell line presenting stable secretion of monoclonal antibody against BAG1. The supenant of the hybridoma cell line was collected, of which the antibody titer and sub-typing were determined by indirect ELISA. Four BALB/c mice were injected intraperitoneally with 0.5 ml liquid paraffin for one week, further intraperitoneal injection was delivered with 106 monoclonal hybridoma cells/mouse. The mice ascites was collected and the monoclonal antibody against BAG1 was purified by affinity chromatography. Lysed protein was prepared from the in vitro induced and in vivo formed ME49 toxoplasma, of which the BAG1 expression was examined by Western blotting using the purified anti-BAG1 monoclonal antibody as the first antibody, and the bradyzoite status was checked by indirect immunofluorescence assay (IFA). Results The open reading frame fragment of bag1 was obtained by RT-PCR, which was 690 bp, encoding 229 amino acids. The recombinant plasmid pET-28a(+)-bag1 was identified by PCR and subsequent sequencing. SDS-PAGE results showed that the relative molecular weight (Mr) of the recombinant BAG1 protein was about 27 000, which was expressed in the form of soluble protein and inclusion body. The results of indirect ELISA showed that the average titer of serum antibody could reach 1 ∶ 102 400 after immunizing mice with purified BAG1 protein for 3 times. Four positive monoclonal hybridoma cell lines were obtained by fusion screening, which were mAb1H03, mAb4B04, mAb5H07, and mAb8B12, respectively. mAb4B04 clone had the highest titer of 1 ∶ 25 600, and the antibody subtype was IgG1. Western blotting showed that the mAb4B04 antibody could detect the BAG1 protein of ME49 strain induced by the alkaline medium in vitro and the BAG1 protein of ME49 strain from mouse brain homogenate. IFA results showed that the mAb4B04 antibody could successfully detect ME49 bradyzoites induced by alkaline medium in vitro and in the BAG1 protein of ME49 strain in the mouse brain homogenate. Conclusion Monoclonal antibodies against T. gondii BAG1 were produced, among them the mAb4B04 antibody with the highest titer can specifically recognize BAG1 protein and bradyzoite of T. gondii ME49 strain.

Analysis of ribosomal and mitochondrial genes of Baylisascaris procyonis
WEI Kai-wen, ZENG Hong-xia, HE Mu, HU Jun-jie
2022, 40(5):  594-602.  doi:10.12140/j.issn.1000-7423.2022.05.005
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Objective To characterize the ribosomal and mitochondrial genes of Baylisascaris procyonis, and their phylogenetic relationship with other Baylisascaris spp. Methods Adult worms of B. procyonis were collected from the intestine of a deceased raccoon at the Kunming Zoo in Kunming, China. After morphological identification, 20 adult worms comprising 10 males and 10 females were selected for morphological measurement and related molecularbiological study. The DNA was extracted from the tissue sample of the parasite using a DNA extraction kit. For amplification of ribosome internal transcribed spacer (ITS), cytochrome c oxidase 1 (cox1), cox2, cox3, cytochrome b (cytb), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), nad5 and tRNA-nad2-tRNA by PCR, which were sequenced and aligned with the database deposited in GenBank using BLAST. Single nucleotide polymorphisms (SNP) were analyzed using MEGA X software, and phylogenetic trees based on ITS, cox1 and cox2 sequences were constructed respectively, using the maximum likelihood (ML) method. Results The adult worms were in ivory color, at a size of (9.15-14.50) cm × (0.20-0.32) cm for females and (6.45-8.70) cm × (0.15-0.20) cm for males. Adult male has copulatory spicule at the caudal ends. The eight genetic markers of B. procyonis were successfully sequenced, and all sequences were deposited in GenBank under the accession numbers MZ092850-MZ092855, MZ164969, MZ172987, MZ172988, MZ172982-MZ172986, MZ172989, MZ172981, and MZ172980 for ITS, cox1, cox2, cox3, cytb, nad1, nad5, and tRNA-nad2-tRNA, respectively. Of the eight loci, the newly obtained sequence shared identities of 98.6%-100%, 98.5%-99.5%, 99.4%-99.7%, 98.3%, 98.8%-99.0%, 98.2%, 97.8% and 99.2%, respectively, with those of B. procyonis previously deposited in GenBank. Additionally, the newly obtained ITS, cox1, cox2 and tRNA-nad2-tRNA sequences had identities of 98.6%-100%, 99.2%-100%, 99.4%-100% and 99.6%-99.9%, respectively, with those of B. columnaris previously deposited in GenBank. SNP analysis showed that the newly obtained ITS, cox1, cox2 and tRNA-nad2-tRNA sequences had 28, 7 and 12 SNP sites, respectively, compared to those of B. procyonis and B. columnaris previously deposited in GenBank. However, none of the SNP sites was suitable to distinguish B. procyonis from B. columnaris. Meanwhile, the newly obtained ITS sequences contained 7 to 9 GA tandem repeats, and the 7 and 8 repeats constituted the first records discovered in B. procyonis. Phylogenetic analysis inferred from ITS, cox1 or cox2 sequences indicated that B. procyonis and B. columnaris obtained from different geographical areas completely aggregated, and formed an individual clade, that clustered with B. devosi. Conclusion The markers sequences and SNP characteristics of B. raccoonus were highly similar to those of B. cylindrica, whereas phylogenetic analysis could not effectively differentiate B. procyonis from B. columnaris.

Prevalence and gene polymorphism analysis of Echinococcus granulosus in cattle and sheep in part areas of Xinjiang
GUO Lu, WU Xiao-xia, DUAN Lan-li, WANG Bing-jie, XU Ning, AREAI Ahatai, WU Yun-hua, ZHAO Li, BAN Wan-li, CHEN Yun-ying, YU Wan-rong, LIU Shuai, PAN Xing-yu, WULIJIANG Kamali, XU Jing, MUNILA Teliewuhan, ZHANG Zhuang-zhi
2022, 40(5):  603-609.  doi:10.12140/j.issn.1000-7423.2022.05.006
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Objective To understand the prevalence and gene polymorphism of Echinococcus granulosus in cattle and sheep in part areas of Xinjiang Uygur Autonomous Region (Xinjiang), and to provide field data for the prevention and control of cystic echinococcosis in this region. Methods From 2020 to 2021, the livers and lungs of the slautered cattle and sheep were collected from the designated slaughterhouses in Yining City, Altay City, Tacheng City, Zhaosu County, Turks County, Emin County and Bole City. The infection was preliminarily assessed by visual inspection and palpation. Suspected E. granulosus cysts were collected for DNA extraction, of which the E. granulosus cytochrome oxidase 1 (cox1) gene was amplified by PCR and sequenced. The sequences obtained were BLAST aligned with the sequences of G1, G3, C4, G5 and G6 downloaded. The phylogenetic tree was constructed by the neighbour-joining method using MEGA 7.0 software, the haplotype network of cox1 gene was constructed by Popart software, and the gene polymorphism sites were analyzed by DnaSPvS software. SPSS17.0 software was used for statistical analysis. The infection rates of cattle, sheep and different organs in different regions were compared by Chi-square (χ2) test. Results A total of 4 977 livestock (563 cattle, 4 414 sheep) were investigated in 7 counties (cities) in Xinjiang, from them 141 cysts were collected. An amplicon of 850 bp was found in 121 cysts, and was successfully sequenced. The overall infection rate in cattle and sheep was 2.07% (103/4 977), among them the highest infection rate in cattle and sheep was seen in Bole City (6.50%, 32/492) compared to Yining City (1.80%, 38/2 108), Tacheng City (1.14%, 9/790), Turks County (1.06%, 2/189) and Emin County (0.72%, 7/970). The difference was statistically significant (χ2 = 49.873, 33.682, 8.762, 28.666, all P < 0.05). The infection rates of cattle and sheep were 3.02% (17/563) and 1.95% (86/4 414), respectively, and the difference was statistically significant (χ2 = 14.296, P < 0.05). The liver and lung cysts from 101 sheep were collected, in which liver cysts accounted for 42.57% (43/101), lung cysts accounted for 27.72% (28/101), and liver and lung co-infection cysts accounted for 29.70% (30/101). The liver and lung cysts from 20 cattle were collected, in which liver cysts accounted for 25.00% (5/20), lung cysts accounted for 45.00% (9/20), and liver and lung co-infection cysts accounted for 30.00% (6/20). BLAST alignment showed that 120 (99.2%) of the 121 sequences had 98%-100% homology with the G1 genotype sequence of E. granulosus (MN886258.1, MG280957.1, KX020359.1 and MG672143.1), which clustered into one branch on the phylogenetic tree, and One (0.8%) had 100% homology with the G3 genotype sequence (MG682532.1), which clustered into one branch on the phylogenetic tree. Haplotype analysis showed that there were 18 haplotypes in cox1 gene sequence, Hap 1-Hap 17 was G1 genotype, Hap 18 was G3 genotype. Hap 1 had the largest haplotype population. There were a total of 20 variation sites among 18 haplotypes. Sequence alignment showed that 52 of 121 sequences had base conversion and transversion. Conclusion The E. granulosus infection rates among cattle and sheep in 7 counties (cities) in Xinjiang are significantly different. G1 (99.2%) and G3 (0.8%) genotypes and 18 haplotypes are found, among which Hap 1 is the dominant pathogenic haplotype.

Genetic diversity and differentiation time of human isolates of Echinococcus granulosus and E. multilocularis from Qinghai
WU De-fang, FU Yong, REN Bin, ZHANG Yao-gang, XU Xiao-lei, PANG Ming-quan, FAN Hai-ning
2022, 40(5):  610-615.  doi:10.12140/j.issn.1000-7423.2022.05.007
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Objective To analyzed the genetic diversity, genetic differences between populations and differentiation time of Echinococcus granulosus and E. multilocularis of Qinghai isolates, in order to provide scientific basis for species tracing and prevention and control of Echinococcus in Qinghai Province, China. Methods For genetic analysis, 50 liver lesion samples were collected from hospitalized echinococcosis patients in the Affiliated Hospital of Qinghai University to extract genomic DNA and amplify mitochondrial dehydrogenase 1 gene (nad1). Sequence multiple alignment was performed using Clustal X v2.0 software. Geographic informatics mapping of patients’ residence was constructed using ArcGIS software. Sequence haplotype analysis was made with DnaSP v6 software. Modeltest 3.7 software and PAUP*4.0B10 software were used to calculate the minimum optimal nucleic acid evolution model. The Bayesian’s phylogenetic evolution tree was constructed with MrBayes-3.2.7 software. The differentiation time of each node in the phylogenetic tree was estimated with the Bayesian method using BEAST v2.6.3 software. Results We successfully identified 48 Echinococcus lesion samples specimen and obtained the full length of complete nad1 gene of 894 bp. Among them, 13 samples were identified as the G1 genotype of E. granulosus, and 35 samples as E. multilocularis. All the sequences showed > 99% similarity to those in GenBank. Four haplotypes were identified as H1-H4 in the two species respectively; H3 was the dominant haplotype in E. granulosus samples(10/13), which is present in Xining, Guoluo, Yushu, Haidong, Haibei and Huangnan. H2 haplotype was found dominant in E. multilocular samples (51.4%,18/35), which is present in Xining, Guoluo, Yushu, and Haidong. The phylogenetic tree showed that E. granulosus and G1 genotype clustered into one branch, and E. multilocularis and Asian strain clustered into one branch. The results of differentiation time showed that the nearest common ancestor of E. granulosus, E. multilocularis, E. vogeli and E. oligarthrus was about 5.5 Mya (95% confidence interval 4.5-6.5 Mya), and the differentiation time of E. granulosus and E. multilocularis was about 2.5 Mya (95% confidence interval 2.3-4.1 Mya). Conclusion Both human E. granulosus and E. multilocularis in Qinghai Province show high genetic diversity. E. granulosus was found of G1 genotype, with H3 as the dominant haplotype, while in E. multilocularis samles H2 is the dominant. The two speies are widely distributed throughout Qinghai Province. The two species of Echinococcus exhit closer genetic relationship and differentiation timing.

Laboratory diagnosis of a rare case of primary amebic meningoencephalitis
CHEN Rui, YUAN Qiong-hui, XIA Wan-bao
2022, 40(5):  616-621.  doi:10.12140/j.issn.1000-7423.2022.05.008
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Objective To report laboratory diagnosis of a rare primary amebic meningoencephalitis case. Methods The clinical data and the cerebrospinal fluid (CPF) sample was collected from the patient, and the CPF sample was smeared and stained with Wright Giemsa for microscopy examination. The genomic DNA was extracted from the CPF for amplification of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 sequence using Naegleria genus- specific and N. fowleri species-specific primers by PCR, and then the amplicon sequenced. The sequence obtained was aligned with sequences in GenBank using BLAST, with which phylogenetic tree was constructed by neighbour-joining method using MEGA5. The CPF sample was sent to Hangzhou Kingdomain Medical Laboratory Co., LTD for DNA-pathogenic microorganism metagenomic detection. Results The patient, a 42-year-old man from Xiangshan, Ningbo, had been bedridden with severe burns covering whole body for 20 years. On July 21, 2022, he was sent to the First People’s Hospital of Xiangshan, Ningbo, Zhejiang Province, for fever, chills, headache, sore throat, nausea and vomiting. The physical examination showed that the weight of the patient was 45 kg, the body temperature was 40.5 ℃, with obnubilation, eyes on the turn, conjunctival congestion. Both eye pupils were 3 mm in diameter, equal in size and circle. The patient also had jaw clenching, neck stiffness, stertorous breathing, limb convulsion, muscle spasm, urinary incontinence, skin and mucous membrane flushing and other clinical symptoms. The head and chest CT showed no obvious abnormalities. The CPF pressure of lumbar puncture was 39 cmH2O (1 cmH2O = 0.098 kPa). Amoeba trophozoites with rapid and continuous amoeba-like movement could be seen on the smear of CPF under microscope. The amoeba trophozoites were also seen after Wright Giemsa staining. The genus- and species-specific gene fragments, which were 183 bp and 311 bp, respectively, were amplified. A total of 21 534 sequences of N. fowleri with a relative abundance of 99.8% were found by DNA-pathogenic microorganism metagenomic testing. No sequence of other pathogenic microorganisms was found. The sequence alignment showed that the amplified ITS1-5.8S-ITS2 sequence was 99% identical to the N. fowleri Na 420c strain sequence (Accession no. AJ132028) recorded in GenBank, and clustered with Amoeba Negri on the phylogenetic tree. The case was confirmed to be infected with N. fowleri. The patient was given meropenem for anti-bacteria, metronidazole for anti-amoeba, fluconazole for anti-fungal treatment, and 20% mannitol dehydration to reduce intracranial pressure. However, due to the rapid deterioration, which lead to respiratory and heart failure, the patient died on July 23, 2022. Conclusion The patient was diagnosed with N. fowleri infection based on the clinical symptoms, pathogenic examination of CPF and molecular biological identification.

Study on anxiety-like behavior of mice with late-stage infection of Schistosoma japonicum
TANG Xian-shi, JI Wen-xiang, XIONG Chun-rong, ZHOU Yong-hua, XU Yong-liang, TONG De-sheng
2022, 40(5):  622-628.  doi:10.12140/j.issn.1000-7423.2022.05.009
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Objective To observe the behavioral characteristics of mice with late-stage schistosomiasis japonica and explore the relationship between late-stage infection of Schistosoma japonicum and anxiety. Methods Thirty 6-week-old male C57BL/6J mice were randomly divided into the infected group (n = 16) and the uninfected group (n = 14). The infected group was infected via abdominal skin contact with 20 cercariae of both sexes, and the uninfected group was treated with dechlorinated water without cercariae. Mice in both of groups were administered with praziquantel by gavage at 8 weeks post-infection. Additional 7 male APP/PS1 double transgenic mice (Alzheimer’s disease model) at 6 weeks of age were used as positive control (transgenic group) without any treatment. Three groups of mice were reared in the same environment until they were 10 months old, to observe their behavioral changes in an open-field test (OFT) and elevated-plus maze test (EPM). In OFT, the total distance, the travelling distance in the central zone, the percentage of travelling distance in the centrol zone, the number of locomotor activity, the duration of locomotor activity, the time percentage spent in the centrol zone, the mean speed, the mean speed in the centrol zone were measured, and in EPM, the locomotor distance, retention time and the number of entry in the open arms/close arms/central zone, and the percentage of locomotor distance, retention time and number of entry in the open arms were recorded to evaluate the anxiety-like behavior of the three mice groups. The mice of all groups were euthanized, and the livers were collected to observe gross morphology. The liver tissue sections were stained using hematoxylin & eosin (HE) and Masson’s trichrome stain for microscopic observation of pathological changes and collagen fiber hyperplasia. The content of liver hydroxyproline which is the specific amino acid constituent in collagen, was determined. The mRNA relative transcription levels of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), collagen Ⅰ(ColⅠ) and ColⅢ in the liver tissues of each group were detected by fluorescence quantitative PCR. SPSS 21.0 software was used for statistical analysis. Results The appaearance color of the mice livers from the infected group was darker compared to the uninfected group. There was a large number of eggs were deposited in the liver tissue, with surrounded infiltrating inflammatory cells and collagen fibres hyperplasia. The liver hydroxyproline content of the infected group (0.194 ± 0.040) μg/mg was significantly higher than that of the uninfected group (0.122 ± 0.016) μg/mg and transgenic group (0.124 ± 0.004) μg/mg (t = 0.79, 0.86, P < 0.05), with no significant difference between the uninfected and transgenic group. The mRNA expression levels of Col Ⅰ, α-SMA, TGF-β1, Col Ⅲ in the liver of the infected group were 3.55 ± 1.76, 2.01 ± 0.66, 2.21 ± 0.64, 4.51 ± 2.32,which is not significantly higher than those of the uninfected group (t = 0.76, 0.59, 0.17, 0.40,P > 0.05). The infected group’s activity time (346.77 ± 181.03) in OFT, the locomotor distance in the open arms (206.31 ± 282.52) mm, the percentage of locomotor distance in the open arms (7.51 ± 9.90)%, the number of entry in the open arms, closed arms and central zone (1.32 ± 1.51, 3.44 ± 3.10, 4.53 ± 4.21)(P < 0.01) in EPM were significantly increased compared to the uninfected group (t = 1.61, 2.24, 1.66, 1.83, 2.35, 2.22, P < 0.05 or P < 0.01), with no significant differences between the two groups for the other indexes. The infected group’s the number of locomotor activity (123.68 ± 50.23) in OFT was significantly decreased compared to the transgenic group (P < 0.05), but there was no significant difference between the two groups for other indexes of OFT. The infected group showed shorter retention time in the closed arm (232.85 ± 88.82) s than that in the transgenic group (295.38 ± 9.88)s(t = -2.81,P < 0.01), and higher values of the rest of the indexes in EPM than those in the transgenic group (P < 0.05 or P < 0.01). Conclusion Mice with late-stage of S. japonica infection did not show more serious level of anxiety than the uninfected mice, however, the anxiety extent presented a significant reducing-tendency, suggesting that the pathological process of S. japonica infection may not be directly correlated to the occurrence and development of anxiety.

Data analysis of clonorchiasis surveillance in high endemic areas of Guangdong Province in 2016—2020
WANG Ke-yi, SHU Huang-fang, FANG Yue-yi, ZENG Qing-sheng, SONG Tie
2022, 40(5):  629-634.  doi:10.12140/j.issn.1000-7423.2022.05.010
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Objective To understand the prevalence of clonorchiasis among people in high endemic areas in Guangdong Province from 2016 to 2020, and to provide a basis for the formulation of prevention and control strategies. Methods For surveillance on clonorchiasis from 2016 to 2020, in Χinhui district of Jiangmen City, Guangdong Province, five regions were designated according to the geographic position in the east, west, south, north and middle part, from each of them, one township (town) was randomly selected as regular surveilence site in Χinhui District of Jiangmen City, Guangdong Province including Da’ao, Sanjiang, Shadui, Siqian, and Yamen. Every year, over 200 permanent residents aged 3 or above were cluster selected from an administrative village of each selected town. Fecal samples were collected from the participants and examined for Clonorchis sinensis eggs using the Kato-Katz thick smear method (two slide readings per sample). Pearson’s chi-squared test was used to compare the rates between groups. Results From 2016 to 2020, a total of 5116 fecal samples were collected and examined from five surveillance sites in Xinhui District, with a overall infection rate of 14.6% (749/5 116). The annual infection rates of C. sinensis from 2016 to 2020 were 23.1% (237/1 025), 15.5% (156/1 005), 13.1% (134/1 021), 13.8% (142/1 031), and 7.7% (80/1 034), respectively, showing a downward trend. The difference in infection rate between different years was statistically significant (χ2 = 101.56,P < 0.01). The infection rates in residents at the five regular surveillance sites were 38.7% (404/1 045) in Da’ao, 13.9% (142/1 025) in Siqian, 10.5% (105/1 003) in Sanjiang, 6.0% (61/1 017) in Shadui and 3.6% (37/1 026) in Yamen. The infection rate in different regions was statistically significant(χ2 = 657.67,P < 0.01). The average age of the infected was 43.1 years old. The lowest infection rate of 5.6% (79/1 415) was seen in the age group of < 18 years, while the highest infection rate of 20.6% (97/47) was seen in the age 61-70 group. The infection rate in different age groups was statistically significant (χ2 = 137.0, P < 0.01). Among all occupations, the highest infection rate of 24.7% (36/146) was seen in public servants. There was a statistically significant difference in the infection rate among different occupations (χ2 = 156.44, P < 0.01). The infection rate among illiterate and semi-illiterate people was 16.8% (17/101). There was no significant difference in the infection rate among people with different education levels(χ2 = 9.33, P > 0.05). The average infection rates in 2016—2020 were 16.5% (412/2 492) and 12.8% (337/2 624) for males and females, respectively. The infection rate in males was significantly higher than that for females (χ2 = 13.93, P < 0.01). Of all the infected individuals, 97.2% (728/749) were infection, 2.8% (21/749) moderately infection, and no severe infections. Conclusion The surveillance conducted from 2016 to 2020 indicated tha C. sinensis infection in residents in the high endemic areas of the Guangdong Province remained considerably high but showed an overall decreasing trend, with mainly having mild infection. Based on the results, future surveillance needs to stress on the males aged over 18 years.

TEACHING RESEARCH
Challenges and development direction of human parasitology teaching in the new era
TAN Bai-hong, WANG Yan-ling, ZHENG Jing-tong
2022, 40(5):  635-641.  doi:10.12140/j.issn.1000-7423.2022.05.011
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With the development of the social economy, the change in life-style, population flow and increasing international travel, the social and natural factors related to the prevalence of parasitic diseases have changed significantly. This study analyzed the new changes in human parasitic diseases and the existing problems of human parasitology teaching in medical colleges. On this basis, several countermeasures were recommended for advancing human parasitology. The purpose of this study is to call on the teaching of human parasitology to keep pace with the times and hope that medical schools can play the role of talent training and basic research and contribute to the prevention, control and diagnosis of parasitic diseases in the new era in China.

REVIEWS
Research advances in Toxoplasma gondii induced host mental-behavioural disorders
DAI Li-sha, ZHANG Li-xin, YIN Kun
2022, 40(5):  642-646.  doi:10.12140/j.issn.1000-7423.2022.05.012
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Toxoplasma gondii can cause zoonotic parasitic diseases in humans and animals. Chronic toxoplasmosis induced by toxoplasma cysts could cause nervous system dysfunction, behavioural changes or mental disorders in the hosts, including reducing the natural fear of predators in the rodent host. Multiple psychiatric disorders in human hosts were also significantly associated with T. gondii chronic infection. The possible mechanisms that regulate this process include cerebral cyst infection, central neurotransmitter disorders, and epigenetic regulation of“behavioural manipulation”, but the precise mechanism remains unclear. In this review, we have summarized relevant studies of the host abnormal mental behaviour induced by T. gondii, and summarized the well-established mechanism(s) for this process.

Research progress on combined use of drugs with albendazole in the treatment of echinococcosis
QIN Min, WANG Li-ying
2022, 40(5):  647-655.  doi:10.12140/j.issn.1000-7423.2022.05.013
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Echinococcosis is a parasitic disease that seriously affects the health and life safety of farmers and herdsmen. Albendazole is the primary clinical drug. However, the bioavailability of albendazole is low, requiring patients to increase the dosage and take medicine for a long time, producing certain adverse reactions and drug resistance. In order to improve the efficacy of albendazole and reduce the damage of drugs to the body, more and more studies pay attention to the combination of albendazole. This paper focuses on the advances progress of other drugs combined with albendazole in the treatment of echinococcosis to provide a reference for the further research and application of drug therapy in patients with echinococcosis.

Research progress on liver fibrosis caused by Echinococcus infection
JIANG Xiao-feng, SHEN Yu-juan
2022, 40(5):  656-660.  doi:10.12140/j.issn.1000-7423.2022.05.014
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Echinococcus infection may cause damage to hepatocytes. Hepatic fibrosis is a process of excessive proliferation of connective tissue after liver injury. In the dynamic process of liver self-healing, the activation of hepatic stellate cells, the increased extracellular matrix production and the decreased extracellular matrix consumption result in fibrosis. This review summarizes the regulatory mechanisms of hepatic stellate cells, cytokines and microRNA involved in hepatic fibrosis caused by Echinococcus infection.

Research advances of multi-epitope vaccine candidates against toxoplasmosis
LI Run-hua, YIN Guo-rong
2022, 40(5):  661-667.  doi:10.12140/j.issn.1000-7423.2022.05.015
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Toxoplasma gondii infects almost all warm-blooded animals causing toxoplasmosis in humans and animals. Despite the significant progress in the recent efforts toward developing an effective vaccine against toxoplasmosis, no human vaccines are available to prevent this disease. However, vaccines based on a single or a few antigen candidates was only able to elicit partial protective immunity against T. gondii infection. Therefore, developing a polyvalent vaccine consisting of antigens from all stages of the parasite life cycle, including tachyzoites, bradyzoites, and sporozoites, is likely to be essential for sterile immunity. The combination of multi-epitopes from antigens of different stages of the parasite life cycle has become an promising strategy for building a potent, safe, and effective vaccine. Epitope-based vaccines have gained attention as novel vaccine candidates due to their ability of inducing protective immune responses. In this review, we have summarized the immunological basis, design, and construction strategy of epitope vaccines. We have reviewed the latest research advances of T. gondii epitope vaccines, and the application prospect of epitope vaccines as a new vaccine development and evaluation strategy.

SHORT COMMUNICATIONS
Clinicial analysis of 12 cases of angiostrongyliasis cantonensis in children
PAN Huo-yun, TAN Li-mei, XU Yi, LI Xu-fang, YE Jia-wei, FANG Chun-xiao, YANG Feng-xia, CHEN Min-xia, YANG Hua-mei, ZENG Fan-sen
2022, 40(5):  668-672.  doi:10.12140/j.issn.1000-7423.2022.05.016
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Retrospective analysis of laboratory tests and imaging examination of 12 children hospitalized for angiostrongyliasis cantonensis infection in Guangzhou Women and Children’s Medical Center from January 2018 to December 2020. Nine cases came from Guangdong, 2 cases came from Guangxi and 1 case came from Yunnan. There were 6 males and 6 females. The age ranged from 1 year and 3 months to 12 years old. There were 4 children with epidemiological histories (1 case of eating river snails, 1 case of eating snails, 1 case had contacted golden apple snail, 1 case of contacting slugs). The most common symptoms were fever (12 cases, all infants have high fever), followed by vomiting (9 cases), and fatigue (7 cases). The peripheral white blood cell count increased in 12 cases [(18.4 ± 4.1) × 109/L], the eosinophil percentage (EOS%) increased in 12 cases [(26.9 ± 10.5)%], the eosinophils absolute count increased in 12 cases [(3.4 ± 2.0) × 109/L]. CSF WBC count increased in 12 cases [(656 ± 424) × 106/L], CSF protein increased in 12 cases [(1.1 ± 0.59) g/L], CSF EOS% increased in 12 cases [(25.7 ± 15.5)%], of which 8 cases were higher than that in peripheral blood. Seven of the 9 children were positive for serum angiostrongyliasis cantonensis antibodies. Three of the 9 children were positive for Angiostrongylus cantonensis antibodies in CSF. Metagenomic next-generation sequencing (mNGS) of the CSF was performed in 11 cases, and Angiostrongylus cantonensis DNA sequence was detected in 10 cases. Imaging examination revealed abnormal head MRI in 8 cases. The main findings were enhancement in leptonmeninges and multiple nodular enhancement in brain. Chest CT showed abnormality in 9 cases, suggesting that ground-glass opacity lesions and multiple small nodular lesions located in the subpleural area were the main manifestations. One child was in coma at admission and died. The other 11 cases were treated with albendazole, and steroids were administrated for 10 cases. The 11 cases were cured after treatment, and there were no sequelae after being followed-up for 2-6 months. The epidemiological history and clinical symptoms of Angiostrongyliasis cantonensis in children were atypical. It should be comprehensively diagnosed by combining laboratory tests, imaging findings, and high-throughput sequencing technology can assist the early diagnosis.

Analysis on the prevalence of Clonorchis sinensis infection in Lingshan County, Guangxi from 2016 to 2019
HUANG Guang-hua, ZHANG Bo, LAI Hai-tao, Lv Guo-li, LIN Yuan, TANG Wen-qian, WAN Xiao-ling, JIANG Zhi-hua, LIU Jian
2022, 40(5):  673-676.  doi:10.12140/j.issn.1000-7423.2022.05.017
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To understand the prevalence of Clonorchis sinensis infection in Lingshan County, one town/village was randomly selected from each of the eastern, western, southern, northern and central administrative regions for investigation from 2016 to 2019. By cluster sampling, 200 permanent residents aged more than 3 years were selected from each study site. More than 1 000 people were surveyed each year with no duplicate enrollment. The fecal samples were collected, and processed using the modified Kato-Katz method (two examinations per sample) for egg counts. Among 4 363 participants, 4.86% (212/4 363) were infected with C. sinensisand the majority were mild infections, accounting for 93.87% (199/212) of the total infections with no severe infection case. The infection rates from 2016 to 2019 were 3.59% (42/1 170), 5.03% (51/1 014), 7.58% (79/1 042) and 3.52% (40/1 137), respectively. The difference between years was statistically significant (χ2 = 25.271, P < 0.01). Among the five survey sites, the highest infection rate was found in Changlu Village, Yandun Town (13.12%, 124/945), and the lowest was found in Chentong Village, Nalong Town (0.37%, 3/821). The difference between study sites was statistically significant (χ2 = 211.912, P < 0.01). The infection rate in males was 7.43% (167/2 248), which was significantly higher than that in females (2.13%, 45/2 115) (χ2 = 66.244, P < 0.01). The 50-59 age group had the highest infection rate (10.4%, 62/596) and the differences between age groups were significant (χ2 = 142.690, P < 0.01). The infection rate of teachers and other occupations was the highest (9.33%, 7/75), and the differences between occupations were significant (χ2 = 81.314, P < 0.01). The highest infection rate found in those with college or higher education levels (8.00%, 2/25), and significant difference in people with different education levels (χ2 = 40.237, P < 0.01). From 2016 to 2019, the prevalence of C. sinensis in Lingshan County was moderate and different for each town/village.

Complete mitochondrial genome sequence of Rhipicephalus microplus
LIU Ya-fang, CHEN Bin, LU Xin-yan, LI Guang-hua, DU Chun-hong, JIANG Dan-dan, YANG Xing
2022, 40(5):  677-681.  doi:10.12140/j.issn.1000-7423.2022.05.018
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Rhipicephalus microplus was collected by the cloth flag method in Yunnan Province in September 2017 and was used for DNA extraction. Four long fragments were amplified by long PCR using the primers designed based on 16S rRNA, 12S rRNA, nad1 and cox3 gene sequences. The fragments were sequenced by conserved primer-walking. The complete mitochondrial genome sequence was obtained by splicing. The length of the mitochondrial genome (mtDNA) of Rhipicephalus microplus is 14 763 bp. It contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding control region of 264 bp (D-Loop region). The base composition was A 38.66%, G 9.02%, C 11.46%, T 40.87%. A + T content was 79.53%. The starting codon of protein-coding genes was ATN. The stop codons were TAA or TAG, except for cox2, cox3, nad5, which used incomplete T as the stop codon. The complete mitochondrial genome have 12 gene overlaps. The length ranges from 1-24 bp. The length of 18 total intergenic regions was 433 bp ranging from 1-311 bp. A total of 22 transferring RNA were found, all of which were typical cloverleaf structures except for tRNACys, tRNAPhe, tRNASer.

Development of PCR diagnostic method for Trichomonas foetus infection based on 18S rRNA
LIU Ji-bing, ZHAO Hong-xi
2022, 40(5):  682-685.  doi:10.12140/j.issn.1000-7423.2022.05.019
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To establish a PCR detection method for Trichomonas foetus, the primers were designed and synthesized according to the 18S rRNA gene sequence of T. foetus published by GenBank. The positive recombinant plasmid pUCm-T-TF18S of T. foetus was used as the template, and the genomic DNA of Giardia felis, Coccidia felis, feline parvovirus and cDNA of feline coronavirus were used as the control for PCR detection to analyze the specificity of this method. The positive T. foetus recombinant plasmid was serial to 8 different concentrations with a gap of 10 folds, and PCR was performed to analyze the sensitivity of this method. The pUCm-T-TF18S plasmids stored at -20 ℃ for 3, 6, 9 and 12 months were detected by PCR to analyze the stability of the method. Twenty cat fecal samples were tested using this established PCR assay and compared with those of microscopic examination. The results showed that the recombinant plasmid pUCm-T-TF18S gave specific bands after PCR amplification. The sequencing results showed that the length of the product sequence was 1 264 bp, and the BLAST sequence comparison analysis showed 99.53% sequence identity, which is consistent with that of T. foetus from cats (GenBank registration number M81842.1). The PCR method for detection of T. foetus had no cross-reactivities with C. felis, G. felis, feline coronavirus and feline parvovirus; the minimum detectable template concentration is 4.52 × 105 copies/μl; The target band of T. foetus DNA can still be detected after being stored in the refrigerator at -20 ℃ for 12 months. This method detected 16 positive samples of T. foetus nucleic acid from 20 cat fecal samples, which is more accurate and sensitive than the results from traditional microscopy (13 samples). It is suggested that the PCR method for the detection of T. foetus is highly specific, sensitive and stable, and can be used for clinical detection and epidemiological investigation of T. foetus.

CASE REPORTS
A case of pulmonary mixed infection of Strongyloides stercoralis and Legionella pneumophila
FU Sha-sha, HAN Chang-yu, WANG Xin-xiao, ZENG Ci-mei, WANG Xiao-qiang, OU Zong-xing
2022, 40(5):  686-688.  doi:10.12140/j.issn.1000-7423.2022.05.020
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A 78-year-old patient was admitted to Haikou People’s Hospital in June 2021 due to “recurrent cough, expectoration and shortness of breath for 9 days and aggravation for 1 day”. The patient is a farmer with a history of frequent soil contact. Wet rales can be heard in both lungsat admission examination. The C-reactive protein was 97.61 mg/L. Sputum culture and blood culture were negative. Chest CT showed multiple mass radiography and exudation in both lungs and a small effusion in the right pleural cavity. A small amount of active bleeding was observed in the upper lobes of both lungs, with a few visible blood clots during bedside tracheoscopy. The patient was initially treated with antibiotics in accordance with the “lung infection” treatment, which did not respond well. Alveolar lavage fluid examination revealed Strongyloides faecalis. Metagenomic sequencing revealed 587 genes of Legionella pneumophila and 155 genes of S. faecalis. Pulmonary strongyliasis and L. pneumophila infection were diagnosed. The patient was treated with albendazole tablets (0.4 g, quaque die, 7 d; 0.4 g, bis in die, 21 d), azithromycin tablets (100 mg, quaque die, 14 d) and levamisole tablets (0.5 g, quaque die, 12 d). The patient’s condition improved 45 days after the treatment. The chest CT showed pulmonary inflammatory lesions absorption improved significantly. One month later, no recurrence was found in the reexamination.

An imported case of ovale malaria in Xinjiang during the epidemic period of COVID-19
SHI Guang-zhong, ZHANG Hai-ting, WANG Shuo, HE Hai-bo, CHEN Xia, MAMATJAN Umar, YU Lin, AYIXIAMU Keyoumu, ZHAO Jiang-shan
2022, 40(5):  689-691.  doi:10.12140/j.issn.1000-7423.2022.05.021
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On December 13, 2020, Yutian County People’s hospital reported one imported malaria case in Hotan, Xinjiang. The patient had worked and lived in Yaounde, Cameroon, from January to September 2020. He was infected with malaria twice in March and May 2020. Antimalarial treatment was administrated by the team doctor for 2-3 days in each treatment. The treatment was stopped after the symptoms improved. The patient returned to China on September 16 and was hospitalized on December 13 due to a high fever of 39℃ and upper respiratory symptoms. Multiple detections of COVID-19 nucleic acid showed negative results. Peripheral blood from the patient was taken for Plasmodium rapid diagnostic test (RDT), which showed a positive result suggesting non Plasmodium falciparum infection. Ring stage P. ovale was found in the blood smear. Nested PCR showed positive for P. ovale. A diagnosis of imported ovale malaria was made. The patient was administrated with 4 dihydroartemisinin piperaquine tablets and 3 primaquine phosphate tablets daily. The malaria parasite test became negative after 8 days of treatment. The patient was followed up for 3 months after discharge and had no symptoms of chills or fever.