A male patient with taeniasis was admitted Tarim University hospital in June 2020. The patient complained that there were milky white peristaltic worm fragments in the feces and the patient reported a history of taeniasis. After administration of areca nuts and pumpkin seeds, one milky tapeworm was expelled. Morphological observation of the expelled worm was carried out under stereo microscope. Combined with epidemiological data and patient’s dietary habits, the sample was preliminarily identified as Taenia saginata infection. The gene amplification results based on the nad1 and rrnS loci of the tapeworm mitochondrion showed that fragments of 529 bp and 357 bp were successfully amplified by PCR. BLAST analysis showed that the gene sequences of the nad1 and rrnS loci had 100% and 99.7% homology to T. saginata collected from human in Kenya (GenBank access number AM503345) and Japan (GenBank access number AB031355). Therefore this tapeworm was identified as T. saginata. The patients were followed up after 3 and 6 months, respectively. The patients were in good health and no tapeworm was expelled.

The pandemic of Corona Virus Disease 2019 (COVID-19) worldwide has prompted the use One Health concept to solve health problems and improve the public health governance system. Using the Superiority Weakness Opportunity Threats (SWOT) analysis method to analyze the opportunities and challenges brought by the current development of One Health in China. The results show that the current advantage is that the Chinese government attaches great importance to One Health, and Chinese scholars are also actively involved in the development of One Health, but there are still disadvantages of weak foundation and low international influence. At the same time, with the opportunity for more recognition of the concept of One Health in the world, China is facing challenges such as insufficient talent competitiveness and unbalanced development in the development of One Health. In this regard, this paper puts forward the strategies and key research contents for developing One Health in China to provide ideas for promoting the development of One Health in China.

Schistosomiasis has been endemic in China for more than 2 000 years, seriously endangering the population’s health and affecting economic development. According to the epidemic characteristics of schistosomiasis, different strategies have been implemented in different historical periods in China to control schistosomiasis. Under the guidance of different strategies, China has achieved remarkable results in schistosomiasis control. The target of schistosomiasis transmission control was achieved nationwide in 2015. The strategies of schistosomiasis control in different periods of China all follow certain scientific basis and rules. This paper reviews the scientific basis of different strategies for schistosomiasis control and puts forward some thoughts and suggestions for schistosomiasis intervention strategies during the 14th Five-Year Plan period to be helpful for the elimination of schistosomiasis.

Objective To identify the main factors affecting the active state of lesions in patients with hepatic cystic echinococcosis and provide the basis for decision-making to improve the diagnosis and treatment of cystic echinococcosis. Methods A total of 251 patients with hepatic cystic echinococcosis receiving surgical treatment at the First Affiliated Hospital of Xinjiang Medical University from between January 2018 and June 2020 were enrolled, and their demographic information and clinical data were collected, Based on the imaging and biological characteristics, the cysts classified as CE1, CE2 and CE3 were grouped as active group, and CE4 and CE5 inactive group. Using the cyst activity as the dependent variable, univariate analysis was performed, based on which unconditinal logistic regression model was constructed to analyze the independent factors influencing the cyst activity status. Results In this study, clinical data of 251 patients with hepatic cystic echinococcosis meeting the inclusion criteria were collected, with 57.0% (143 cases) female and 43.0% (108 cases) male. The age of the patients ranged from 4 to 85 years old, and the average age was (41.31 ± 15.05) years old. Most of the patients had a low educational level, with high school or below accounting for 79.3% (199 cases); the rural resident accounted for 66.9% (168 cases), which is higher than that of the urban resident 33.1% (83 cases). The percentage of CE in Han nationality was 42.2% (106 cases), while that in other minority nationalities was 57.8% (145 cases). The percentage of patients with their first infection was 78.1% (196 cases), which is higher than that of the recurrent infection (21.9%, 55 cases). The active lesions accounted for 76.9% (193 cases), and the inactive lesions accounted for 23.1% (58 cases). The results of the univariate analysis showed that there were significant differences among patients with different age groups, ethnic groups, the active state of lesions between patients with hepatic cystic echinococcosis and those without recurrence, the number of lesions, the size of lesions, red blood cell count, prothrombin time, direct bilirubin and alkaline phosphatase (χ² = 17.110, 7.797, 2.906, 4.702, 16.520, Z = -1.989, 2.446, 2.003, 1.914, P < 0.1). Unconditional Logistic regression results showed that the main factors affecting the active status of lesions in patients with hepatic cystic echinococcosis were age and lesion size. Larger lesions tend to occur in younger patients(P < 0.05). Conclusion The analysis on the clinical data of 251 hepatic cystic echinococcosis patients receiving surgical treatment demonstrated that younger age and larger cyst size were the independent factors affecting the activity status of cysts.

Malaria is an endemic infectious disease caused by Plasmodium and is mainly prevalent in tropical and subtropical areas. Despite the World Health Organization’s announcement of the country’s malaria elimination certification in 2021, the threat of imported malaria will persist as international travel increases. In order to facilitate clinicians’ understanding and rational treatment of malaria and to improve the diagnosis and treatment of malaria, we have invited experts in the relevant field of infectious diseases and parasitic diseases in China to prepare the guidelines for malaria diagnosis and treatment. The guidelines introduce the etiology, epidemiology, pathogenesis, clinical manifestations, diagnosis and differential diagnosis, treatment, care, and prevention of malaria, emphasising on treatment options for different clinical conditions, so that the clinician can use them properly.

Objective To analyze the endemic situation and epidemiological characteristics of echinococcosis in China, and provide a scientific basis for optimizing control strategy. Methods From the Infectious Diseases Report System of the Chinese Center for Disease Control and Prevention, information of nationwide echinococcosis cases reported in 2004 to 2020 were collected, and descriptively analyzed on time distribution, the regional distribution, and demographic distribution, using Microsoft Office 2016 software. Results From 2004 to 2020, a total of 66 040 cases of echinococcosis were reported in 31 provinces (municipalities and autonomous regions), among which 65 340 cases were reported in nine endemic provinces (Xinjiang, Sichuan, Qinghai, Gansu, Ningxia, Tibet, Inner Mongolia, Shaanxi and Yunnan) and 700 cases were reported in 22 non-endemic provinces, accounting for 98.9% and 1.1% of the total, respectively. Among the nationwide total, 31 494 male cases and 34 546 female cases were recorded. The toal number of reported cases of echinococcosis was on the increase from 991 in 2004 to 3 650 on 2020, showing an increasing trend (z = 2.27, P < 0.05). The number of reported cases of echinococcosis from non-endemic provinces in 2004 was 17 cases, which increased to 56 in 2020, showing an increasing trend (z = 4.1675, P < 0.05). The age distribution indicated that majority (91.3%) of cases were at the age goup of ≥ 20, whereas 11 cases were at the age group of under one year old, and 200 cases were at 85 years old or older. The occupation distribution demonstrated that herdsmen and farmers accounted for 72.8% (48 074/66 040) of the total, housework and unemployed 5.5% for (3 606/66 040); and students for 4.6% (3 034/66 040). A total of 56 cases were reported in non-epidemic areas in 2020, including 38 imported cases. Henan reported the most cases with 16, Xinjiang imported 24 cases, and 10 provinces had suspected local cases. Conclusion The endemicity of echinococcosis in nine west provinces remains high. The number of reported cases showed increasing trend. Herdsmen, farmerd, youth and middle age are the population with high prevalence.

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.

A 58-year-old male patient was admitted to the Affiliated Hospital of Xiangnan University in September 2021. He complained of a right neck mass accompanied by pain for 1 week and had a long history of having raw salmon and raw pickled shrimp diet. Physical examination showed a soft neck, midline trachea, and no palpable thyroid mass on both sides. The palpable right sternocleidomastoid muscle had a size of approximately 5 cm × 5 cm × 3 cm, medium texture, regular shape, unclear boundary, poor mobility, no movement with swallowing, and multiple enlarged lymph nodes were palpable in the right neck and supraclavicular fossa, the largest was about 2 cm × 1 cm, with medium texture and good mobility. Blood cell count showed eosinophil count 2.1 × 10⁹/L (0.02 × 10⁹/L-0.52 × 10⁹/L), eosinophil percentage 10.4% (0.4%-8%). Routine stool examination showed no obvious abnormality. Serum IgG antibody was positive. Colour ultrasound of the neck showed multiple cystic, solid mixed echo areas in the right sternocleidomastoid muscle, with a range of about 60 mm × 25 mm × 53 mm, irregular shape, unclear boundary and uneven internal echo. The capsule of the tumour is intact, there seems to be a coiled cord inside, and the blood supply around the capsule is rich. Contrast-enhanced CT of the neck showed that the lower end of the right sternocleidomastoid muscle was significantly enlarged with uneven density, with multiple low-density areas within it, and annular enhancement was observed in the parenchyma during enhancement. Intraoperative exploration revealed that the right sternocleidomastoid muscle mass had a complete capsule. After the incision, clear fluid flowed out of the capsule accompanied by 3 white parasites slowly peristalsis. The parasite body is milky white, long band, the head segment is small, spoon-shaped, the parasite body is relatively large, its dorsal and ventral surface each has a narrow and deep concave suction groove, the front end of the body is depressed and slightly larger, the body does not segment but has transverse folds, the tail is thin. Combined with the morphological identification and epidemiological history of the patients, the infection was considered as Diphyllobothrium latum infection. After the operation, praziquantel (total dose 120 mg/kg, divided into 5 days, three times a day) was treated with deworming, and no abnormality was found in follow-up 3 months later.

Globally, most strains of Cryptosporidium parvum subtypes cause zoonotic infections. Dairy cattle are the important host for the natural infection of C. parvum and the important source of infection for human cryptosporidiosis. In this paper, the molecular epidemiology of C. parvum from dairy cattle was reviewed. The Ⅱd subtype distribution of C. parvum in dairy cattle in China was analyzed. The diversity distribution of Ⅱd subtype was summarized and the development trend of Ⅱd subtype was predicted. This review is of great significance for the prevention and control of C. parvum.

Malaria epidemiological data in 31 provinces/municipalities/autonomous regions (excluding Taiwan region, Hong Kong SAR and Macao SAR) of China in 2022 were collected from the Information System for Parasitic Diseases Prevention and Control. The epidemiological characteristic of malaria cases was analyzed. In 2022, a total of 845 malaria cases were reported in China, which is increased by 5.8% compared to that in 2021 (799 cases). Of all these cases, 844 were imported cases and 1 was local recurrent case infected with Plasmodium malariae with along incubation period. In addition, 820 cases were of Chinese nationality (97.0%, 820/845) and 25 cases were of foreign nationality (3.0%, 25/845). Most of the cases were within the age range of 50-59 years (29.5%, 249/845), with a male-to-female ratio of 17.0:1. The reported cases included 494 cases of P. falciparum infection (58.8%, 494/845), 204 cases of P. vivax infection (24.1%, 204/845), 108 cases of P. ovale infection (12.8%, 108/845), 31 cases of P. malariae infection (3.7%, 31/845), and 8 cases of mixed-infection (0.9%, 8/845). The cases were reported from 26 provinces/municipalities/autonomous regions, with the top 5 provinces including Guangdong (182 cases), Yunnan (136 cases), Sichuan (72 cases), Zhejiang (64 cases) and Henan (59 cases), from which 513 cases (60.7%, 513/845) were reported. A total of 36 severe malaria cases (4.3%, 36/845) and 6 deaths (0.7%, 6/845) were reported. Although there has been no report of indigenous malaria cases in China for six consecutive years, there is still a risk of cluster outbreak of imported malaria and reemerging. After malaria elimination, malaria surveillance and response should be further strengthened, and malaria cases should be detected timely with accurate, diagnosis and standard treatment, so as to reduce the severe cases and deaths and finally prevent the reemerging of malaria transmission.

Malaria is one of the most important infectious diseases in the world and seriously threatens human health. It remains a critical problem to interrupt malaria transmission. Plasmodium parasites invade and multiply inside the host cells causing severe pathological damage to the host. As the interface between the host cells and Plasmodium parasites, PVM is of crucial importance for the survival of Plasmodium parasites. This manuscript briefly introduces the formation of PVM and its relationship with PPM and TVN. The PVM-associated proteins and their functions in blood-stage, liver-stage and sexual-stage parasites are summarized. This review also presents perspectives on malaria control strategies.

Zoonotic diseases are not only significant risks to humans, but also seriously affect global biodiversity conservation. The eastern Tibetan plateau has the highest echinococcosis prevalence in the world. The complex and stable echinococcosis transmission networks is based on several wild and domestic animal species. In this study, we summarized the epidemiological research advances of echinococcosis in the rural area of the Tibetan plateau. We also discussed the ecological factors and their functional mechanisms for the transmission of echinococcosis including host species community composition and dynamics, host species behaviour ecology, and human disturbance. Based on these discussions, we put forward suggestions for a better integrative strategy for echinococcosis control and the sustainable development of the plateau ecosystem.

An 42-year-old male patient who was found to have a parasite in his ileocecal region during a colonoscopy examination in December 2021. The worm was reported motile and was removed with biopsy forceps. During the colonoscopy, no abnormality was found in the intestinal mucosa where the parasite was found and no abnormality was found in the intestinal lumen. The extracted parasite was identified as Anisakis simplex preliminarily by morphological identification. DNA was extracted from parasites and tested by PCR. The amplified fragment was about 1 000 bp. After sequencing, BLAST analysis showed that it was consistent with Anisakis simplex up to 99%. By phylogenetic tree analysis, it was clustered with Anisakis simplex. The parasite was confirmed to be Anisakis simplex. The patient reported that he had lived in southeast coastal cities for a long time and had a history of eating raw seafood products for many years. No discomfort was reported after the parsite was removed, so no other examination and treatments were performed.

Plasmodium vivax is the most geographically widespread malaria parasite that causes most malaria infection cases outside the most malarious continent, sub-Saharan Africa. P. vivax preferentially invades reticulocytes, with a high species specificity. P. vivax reticulocyte binding protein (PvRBP) family has been implicated in roles in reticulocyte invasion and severity of P. vivax infections. The members of PvRBP family act as invasion ligands that mediate new pathways for the parasites to invade reticulocytes, and therefore, are considered important immune targets. Importantly, PvRBP2a-CD98 and PvRBP2b-TfR1 have been identified as two major ligand-receptor pairs implicated in the invasion of reticulocytes by P. vivax. The Pvrbp family is highly polymorphic and can generate immune evasion, which can increase the efficiency of vivax malaria invasion and the severity of the disease. With the advances in research on the molecular mechanisms of P. vivax invasion, insights have been provided on members of this protein family as promising antimalarial vaccine candidates, able to generate high titer antibodies for effective prevention and control of vivax malaria. This review summarizes the role of reticulocytes in P. vivax infection and the function of the PvRBP proteins family as immune targets in the human population.

Objective To investigate the effect of osteopontin (OPN) expression level on the growth and development of Echinococcus multilocularis protoscoleces. Methods Protoscoleces were isolated from E. multilocularis-infected Meriones unguiculatus and cultured in vitro for 3 days. The E. multilocularis protoscoleces were assigned into four groups: LV-EmOPN-734 (knockdown EmOPN) group, LV3-NC (knockdown control) group, LV-EmOPN-0423 (overexpression EmOPN) group, and LV5-NC (overexpression control) group. Each group was tested in parallel triplicate wells, with 5 000 protoscoleces in each well. LV-EmOPN-734 lentivirus diluent (final concentration 1.4 × 10⁷ TU/ml) was added to the silenced EmOPN group, LV-EmOPN-0423 lentivirus diluent (final concentration 4.83 × 10⁸ copies/ml) was added to the EmOPN overexpression group, and the corresponding control group was added with the same amount of lentivirus diluent (blank plasmid). After 72 hours of lentivirus infection, the fluorescence intensity of each group of protoscoleces was observed under fluorescence microscope, the relative expression of OPN in protoscoleces was detected by Western blotting, Caspase-3 activity was detected by applying cysteine aspartate-3 (Caspase-3) assay kit, and the proliferation ability of protoscoleces was detected by applying EdU Imaging kit (Cy3). 72 hours after infection, the protosegment was co-cultured with rat hepatoma cells for 8-12 weeks, and the morphology of the germinal layer of E. multilocularis vesicles was observed by scanning electron microscopy. Statistical analysis of differences between groups using t-test. Results After 72 hours of lentivirus infection, it can be seen that the E. multilocularis protoscoleces were infected by lentivirus successfully, which is distributed in a circle or dots. Western blotting results showed that the relative expression of EmOPN in the LV-EmOPN-734 group was (0.43 ± 0.04), which was lower than that in LV3-NC group (0.80 ± 0.07) (t = 8.623, P < 0.01); the relative expression of EmOPN in LV-EmOPN-0423 group was (1.18 ± 0.21), which was higher than that in the LV5-NC group (0.73 ± 0.06) (t = 3.333, P < 0.05). Caspase-3 activity assay showed that the Caspase-3 activity was (61.55 ± 1.64) μmol/L in the LV-EmOPN-734 group, higher than (28.20 ± 2.16) μmol/L in the LV3-NC group (t = 24.57, P < 0.01); Caspase-3 activity in the LV-EmOPN-0423 group was (50.11 ± 6.45) μmol/L, which was lower than (78.22 ± 16.43) μmol/L in the LV5-NC group (t = 3.185, P < 0.01). The results of EdU Imaging Kits (Cy3) assay showed that the protoscoleces of EdU+ in the LV-EmOPN-734 group were(0.47 ± 0.06), which was lower than that of the LV3-NC group (0.72 ± 0.10) (t = 3.663, P < 0.05); the protoscoleces of EdU+ in the LV-EmOPN-0423 group had of EdU+ protoscolecesof (0.81 ± 0.09), which was higher than that of the LV5-NC group (0.54 ± 0.06) (t = 4.309, P < 0.05). Scanning electron microscopy observations showed that most of the germinal layer cells of the LV-EmOPN-734 group were collapsed, shrank, shed to a large extent, and lost their normal structure; while the germinal layer cells of the LV-EmOPN-0423 group had intact cell membranes and full morphology, forming germinal layer vesicles and growing small tips to connect with the germinal layer. Conclusion The OPN expression level upon rising-up or lowering-down may promote or inhibit the growth and development of the E. multilocularis protoscoleces.

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.

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.

A 64-year-old female patient, who is a farmer, was admitted to the hospital for intermittent pain and discomfort in the lower back. The patient was positive for human immunodeficiency virus (HIV) antigen/antibody test. Blastocystis hominis and Giardia lamblia flagellate cysts were found by microscopic examination for the normal saline smear of the fecal sample. The B. hominis cysts were round, oval, and varying in size, with 1 to 2 granular nuclei. Vacuoles were seen in the middle of the iodine-stained parasites with irregular, shiny, crescent-like edges between the vacuoles and the cell membrane. Many small bright spots were seen in the vacuoles. The G. lamblia flagellate cysts are oval in shape, with thick cyst walls. There is a clear gap between the cyst and the parasite. The iodine stains the inner axis of the cyst with 2 to 4 nuclei. The nuclei are located on both sides of the axis. Four purplish red nuclei were evident in the cyst after hematoxylin staining. Nested PCR amplified a 511 bp band from patient fecal DNA, which was 99% identical to the sequence TSA417 of G. lamblia (GenBank accession number AF065606). The ribosomal small subunit gene was amplified by PCR with the specific primers of BhRDr and RD5 of B. hominis, and a band of approximately 600 bp was amplified. The sequencing suggested it was B. hominis ST1 type. Combined with the patient’s epidemiological history of drinking unboiled water, using dry-toilet, breading cats and dogs, etc., the diagnosis of HIV co-infection with B. hominis and G. lamblia was made. After 4 days of treatment with metronidazole (0.2 g/time, 3 times/d), the patient’s symptoms were relieved, and the patient was discharged.

Objective To establish and preliminarily evaluate a fast, convenient and visualized method for detection of Schistosoma mansoni nucleic acid by combining the recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD). Methods Selecting the cytochrome c oxidase subunit 1 (COX1) gene of S. mansoni as the target sequence, specific primers and probes were designed using Primer Primer5 with manual assistance and screened to establish a LFD-RPA method for detection of S. mansoni. The sensitivity of the established method was evaluated by detecting the genomic DNA of adult S. mansoni at different concentrations (1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, 1 fg/μl, 0.1 fg/μl) and the recombinant SmCox1 plasmids with different copies (10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10^-1 copies/μl). The specificity of LFD-RPA was evaluated by detecting the genomic DNA of S. japonicum adult worms, S. haematobium eggs, S. mansoni infected and non-infected Biomphalaria spp., S. japonicum infected and non-infected Oncomelania hupensis and other trematodes. Thirty female BALB/c mice were randomly divided into low infection group (40 cercariae per mouse), high infection group (80 cercariae per mouse) and control group (no infection) to establish the mouse infection model. The DNA was extracted from the feces and serum samples from the mice at different time points post infection (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks) and detected by LFD-RPA to evaluate its effectiveness in early detection of S. mansoni infection. Results The specific band of S. mansoni could be detected by the established SmCox1-LFD-RPA method after 20 min of reaction at 39 ℃. The detectable limits of LFD-RPA method for S. mansoni were 10 fg/μl for the genomic DNA of adult worms and 10 copies/μl for SmCox1 recombinant plasmid, respectively. The established SmCox1-LFD-RPA method was specific to the DNA of S. mansoni and S. mansoni infected Biomphalaria spp., no cross-reaction with other flukes were observed. When detecting DNA samples extracted from mouse blood and feces from the 40 cercariae group 1 week to 8 weeks post infection, SmCox1-LFD-RPA presented positive reaction since 3 weeks post infection. While for the mice of the 80 cercariae group, positive band began to appear from 1-week post infection. The color of the band was gradually deepened with the infection time. Conclusion A LFD-RPA based visualized rapid detection method for S. mansoni nucleic acid was developed, with higher sensitivity, specificity, rapidity and easy-to-use.

Objective To establish a reverse transcription-enzymatic recombinase isothermal amplification (RT-ERA) assay for detection of specific gene segment of West Nile virus. Methods The highly conserved region of NS5 gene (120 bp) was selected as the target gene fragment to be detected. The primers and fluorescence probes were designed and synthesized based on the isothermal amplification principle to establish a fluorescence RT-ERA assay system. The fluorescence RT-ERA assay was performed to detect serial diluted recombinant plasmids (10⁴, 10³, 10², 10, 1 copy/μl) containing target gene fragments and West Nile virus RNA at different concentrations (10, 1, 10^-1, 10^-2, 10^-3 ng/μl) to determine the sensitivity. Further, this assay was applied to detect the genomic RNA of Tick-Borne Encephalitis virus, Chikungunya virus, Yellow fever virus, Dengue virus type I, Japanese Encephalitis virus and Influenza A virus (H2N1) to evaluate the specificity. Results A fluorescence RT-ERA assay was successfully established, which was effective in amplifying the specific gene fragments of West Nile virus within 20 min at 39 ℃. The minimum detectable limit of the fluorescence RT-ERA assay was 1 copy/μl using recombinant plasmids as templates and 10^-3 ng/μl using West Nile virus RNA samples as templates. The results of the fluorescent RT-ERA assays were all negative for detecting the genomic RNA from tick-borne encephalitis virus, chikungunya virus, yellow fever virus, dengue virus type I, Japanese encephalitis virus, and Influenza A virus (H2N1). Conclusion A fluorescence RT-ERA assay for detection of West Nile virus RNA is successfully established, which is easy to use, sensitive and specific.