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    30 August 2016, Volume 34 Issue 4
    Resistance of rAAV2-MfE77.43-Transferred Mice to Schistosoma japonicum Infection
    WANG Hong-mei1, XIONG De-hui2, LUO Sai-qun2, YANG Jie1, QIAN Yin-jun3, QIN Zhi-qiang3*
    2016, 34(4):  1-297-302. 
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    Objective To investigate the resistance of E77.43 gene of Microtus fortis(MfE77.43) to Schistosoma japonicum infection. Methods MfE77.43 was constructed into the recombinant Adeno-associated virus AAV2. The AAV2-MfE77.43 was transfected into HEK293 cells by the calcium phosphate DNA coprecipitation method. The recombinant rAAV2-MfE77.43 was purified and total RNA was extracted from the transfected cells. The expression of E77.43 was examined by RT-PCR and the purity of rAAV2-MfE77.43 was analyzed by SDS-PAGE. Eighteen KM mice were divided into three groups (n=6 in each group). Mice in the experiment group were intramuscularly injected on days 0, 3 and 7 with 400 μl recombinant AAV2-MfE77.43 virus which was 5-fold diluted in normal saline. Mice in negative control and blank control groups received same volume of pAAV or normal saline. Venous blood was collected through the tail before each injection, and E77.43 expression in plasma was detected by dot-ELISA method. After the last injection, each mouse was infected with 40 S. japonicum cercariae and sacrificed on day 41 after infection. Adult worms and liver eggs per gram(LEPG) were counted. Worm and egg reduction rate was calculated respectively. Egg granulomas were observed by HE staining. Results RT-PCR resulted in a 330 bp specific band. SDS-PAGE of virus shell protein revealed three protein bands with Mr of 87 000, 72 000 and 62 000, respectively. Dot-ELISA showed that E77.43 protein began to be expressed on day 3 after rAAV2-MfE77.43 injection, remaining stable till day 41. The adult worm number and LEPG were 20.16±3.93 and 19 800±2 715, respectively, with a worm and egg reduction rate of 27.3% and 26.2% in the experiment group. While the worm number and LEPG in the negative control group were 29.16±2.44 and 28 000±2 192(P<0.01), respectively. HE staining and observation revealed fewer eosinophils and inflammatory cells around the liver eggs in the therapy group. Conclusion The E77.43 gene shows protective effects against S. japonicum infection, indicating that E77.43 may participate in the natural resistance of Microtus fortis to S. japonicum infection.

    Establishment of Magnetic Affinity Enzyme Linked Immunoassay Based on Schistosoma japonicum Recombinant Antigen Sj26 and Its Application in Detection of Serum Antibody with Low Intensity of Infection
    YU Qin1*, ZHU Yan-hong2, GUAN Fei3, YANG Hai2
    2016, 34(4):  2-303-307. 
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    Objective To develop the magnetic affinity enzyme-linked immunoassay(MEIA) using recombinant glutathione-S-transferase of Schistosoma japonicum with a relative molecular weight of 26 000(rSj26-MEIA) for antibody detection under low intensity infection. Methods The recombinant plasmid pET28a-Sj26 was transformed into Escherichia coli strain BL21, and 0.6 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce its expression. The expression products were purified by Ni2+ (nickel sulfate) affinity chromatography, SDS-PAGE and Western blotting were performed to examine the expression of rSj26. The purified rSj26 was coupled to magnetic beads as capture antigen and the reaction conditions were optimized to establish the rSj26-MEIA method. The method was then used to analyze 58 serum samples from patients with low-intensity S. japonicum infection, 30 serum samples from non-endemic areas as a negative control, and 6 serum samples from patients with paragonimus infection. Results were compared with those obtained with ELISA. Results The concentration of purified rSj26 was 2.5 mg/ml. The rSj26 had a relative molecular weight of 27 000 and was expressed mainly in the soluble form as revealed by SDS-PAGE. It could be recognized by rabbit and murine sera infected with S. japonicum as shown by Western blotting. Optimization of rSj26-MEIA revealed that use of 0.2 mg magnetic beads loaded with 10 μg rSj26 and serum sample dilution at 1 ∶ 100 yielded the highest ratio of the mean A550 of positive serum to the mean A550 of negative sample(P/N)(3.97). For serum samples from patients with low-intensity S. japonicum infection, rSj26-MEIA and rSj26-ELISA both resulted in a positive detection rate of 24.14%(14/58), and P/N values of 3.61 and 2.56. In addition, Pearson′s correlation analysis revealed positive correlation between A550 values detected by rSj26-MEIA and by rSj26-ELISA(r=0.658, P<0.01). Further, no positive reaction was found in the 6 serum samples from patients with paragonimus infection and in the 30 serum samples from non-endemic areas, either by rSj26-MEIA or by rSj26-ELISA. Conclusion rSj26-MEIA may be used as a new technique for detection of serum antibody against S. japonicum infection with low intensity.
      
    The Molluscicidal Effect of Chlorosalicylicamide Sustained-release Granules on Oncomelania hupensis
    WEI Feng-hua1*, QIN Jie2, LI Bo1, LIU Min2, HE Hui1, LI Xiao-ping1, YUAN Yi1,HE Zheng-wen3, HUANG Wen-jun3, XU Zhao-gang3, JI Jie4, YANG Ji-xing4,WANG Zeng-zhen5, LI Gui-ling2, GONG Xin-guo1, XU Xing-
    2016, 34(4):  3-308-314. 
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    Objective To evaluate the molluscicidal effect of the chlorosalicylicamide sustained-release granules(LDS-SRG) on Oncomelania hupensis. Methods Seven effective concentrations or dosages of LDS-SRG, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 mg/L (for immersion test) or g/m2(for spraying test), were prepared from the original 5% and 10% concentrations or dosages in the laboratory. In the immersion test, each concentration of LDS-SRG was incubated with 3 packs of snails(30 snails in each pack), and each pack was taken for snail counting at 24, 48 and 72 h respectively. In the spraying test, each dosage of LDS-SRG was applied to 200 snails, and the snail mortality was calculated in 50 randmoly collected snails on days 3 and 7, and in the whole on day 14 after administration. In the field immersion test, LDS-SRG at concentrations of 0.4, 0.8 and 1.6 g/m3 was incubated with 6 packs of snails (30 snails in each pack), and each 2 packs were taken at 24, 48, and 72 h to calculate the snail mortality. In the field spraying test, 0.8, 1.6 and 3.2 g/m2 LDS-SRG was sprayed in 3 snail-positive ditches (~100 m2), and 10 boxes of snails were selected in each ditch on days 3, 7 and 14 to calculate the snail mortality. The 50% wettable powder of niclosamide ethanolamine salt (WPN) with effective concentrations or dosages of 1.0 mg/L (or g/m2 and g/m3) was used as the positive control. Fresh water served as the blank control. Results In the  labratory immersion test using the original concentration of 5%, both 0.1-6.4 mg/L LDS-SRG for 72 h and 1.6-6.4 mg/L LDS-SRG for 48 h caused 100% mortality; and the concentration lethal to 50% (LC50) at 24, 48 and 72 h was 0.70, 0.01 and 0.01 mg/L respectively. When using the original concentration of 10%, both 0.1-6.4 mg/L LDS-SRG for 72 h and 0.2-6.4 mg/L LDS-SRG for 48 h caused 100% mortality; and the LC50 at 24, 48 and 72 h was 0.15, 0.01 and 0.01 mg/L respectively. The labratory spraying test showed that 7-day administration of 1.6 and 6.4 g/m2 LDS-SRG as well as 14-day administration of 3.2 and 6.4 g/m2 LDS-SRG prepared from 5% dosage, resulted in a snail mortality>95%, with the LD50 on days 3, 7 and 14 being 0.06, 0.16, and 0.18 g/m2; 14-day administration of 1.6 g/m2 LDS-SRG as well as 7-day administration of 6.4 g/m2 LDS-SRG prepared from 10% dosage, resulted in a snail mortality>95%, with the LD50 on days 3, 7 and 14 being 3.29, 0.75, and 0.16 g/m2. The mortality by various dosages of LDS-SRG prepared from 5% dosage was significantly higher than that of the control group(P<0.05). In the field immersion test, the snail mortality by 1.6 g/m3 LDS-SRG prepared from 5% and 10% concentrations for 72 h was 96.43% and 98.21% respectively(P>0.05 versus the control group). In the field spraying test, the snail mortality by 3.2 g/m2 LDS-SRG prepared from 5% dosage for 3, 7 and 14 days was 93.99%, 91.18% and 86.48% respectively, and that from 10% dosage was 94.95%, 93.50% and 85.43%, all significantly higher than that of the control group (82.83%, 72.38% and 48.38%)(P<0.05); the snail mortality by 0.8 g/m2 LDS-SRG prepared from 5% dosage for 14 days(66.51%) and that by 1.6 g/m2 LDS-SRG prepared from 5% dosage for 3 days(84.61%) were both significantly higher than that by 10% LDS-SRG(20.13% and 43.06%) (P<0.05). Conclusion The 5% and 10% LDS-SRG used separately in the immersion test and the spraying test both meet the requirements of the national standard of Efficacy Test Methods and Evaluation of Molluscicide for Pesticide Registration.

    Changes of Phenotype and Phagocytosis of Peritoneal Macrophages in Mice Infected with the Larval-stage of Echinococcus granulosus
    PAN Wei1,2, ZHANG Yu-mei2,3, SUN Fen-fen4, CAO Jian-ping2, SHEN Yu-juan2*
    2016, 34(4):  4-315-318. 
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    Objective To investigate the phenotype and phagocytosis changes of the peritoneal macrophages (Mφ) in mice infected with the larval-stage Echinococcus granulosus, and explore the role of Mφ in the responses to parasite infection. Methods Twenty-four female BALB/c mice(age of 6-8 weeks) were randomly assigned into control group and infection group(n=12 in each group). The mice in the infection group were intraperitoneally injected with 2 000 protoscoleces, while the control mice were injected with equal volume of PBS. Five months after infection, the peritoneal mononuclear cells were collected, and the percentage of Mφ and the expression of surface markers CD40, CD80, CD86, and major histocompatibility complex Ⅱ(MHCⅡ) were determined by flow cytometry. The absorbance(A490 value) of Mφ at different concentrations(1×106, 5×105, 1×105) was determined by the neutral red assay to evaluate the phagocytic ability of Mφ. Results The Mφ constituted(30.40±3.15)% and(20.75±5.91)% in mononuclear cells in the infection and the control groups, respectively. The percentages of Mφ expressing CD40, CD80, CD86, and MHC Ⅱ were(45.33±5.51)%, (61.00±10.61)%, (56.88±10.66)% and (27.00±3.82)% in the infection group, which were all significantly higher than those in the control [(41.43±6.19)%, (59.23±8.65)%, (10.91±1.82)% and (13.67±3.01%)] (P<0.05). The A490 values of Mφ at 1×106, 5×105, 1×105 were 0.41±0.03, 0.24±0.05 and 0.16±0.01 in the infection group, which were significantly lower than those in the control (0.61±0.15, 0.47±0.07 and 0.18±0.01)(P<0.01). Conclusion The phagocytic ability of peritoneal Mφ is dramatically weakened after infection, but the expression of activation-associated surface markers is significantly up-regulated after infection.

    Epidemiological Survey on Echinococcosis in Wenquan County of Xinjiang in 2009-2014
    Polat IMINJIAN1, Xamixidin1, LIU Ming-tao1, Yisilayin OSMAN2*
    2016, 34(4):  5-319-323. 
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    Objective To understand the prevalence and distribution of echinococcosis in Wenquan County of Xinjiang, and provide evidence for control of this disease. Methods In 2009-2014, B ultrasound scan of the abdomen was conducted to examine hydatid cyst infection in populations randomly selected in 74 villages in Wenquan County. ELISA was performed to detect serum IgG antibody against hydatid cyst in 6-12-year-old students. Visceral necropsy was performed to examine hydatid cyst infection in livestocks at slaughter house. Dog feces were collected, and hydatid cyst antigen was examined by ELISA. Results A total of 49 627 people were examined, with an average age of 36 years (5-89 years). Among them, 192 (0.39%) were determined to be positive for cystic echinococcosis. The morbidity showed a trend of decrease during 2009-2014, highest in 2009(1.22%, 72/5 919) and lowest in 2014 (0.17%, 15/9 011). There was no significant difference in morbidity between males (0.34%, 72/21 226) and females(0.42%, 120/28 401). The disease was mainly distributed in age range of 20-59 years (91.67%, 176/192). There was no significant difference on morbidity in agricultural areas(0.43%, 130/30 492), pastoral areas (0.33%, 49/14 649) and towns (0.29%, 13/4 486). The morbidity in herdsmen (0.34%, 86/24 963) was significantly higher than that in those with other occupations. The morbidity was higher in Mongolian(0.51%, 45/8 696) and Kazak (0.42%, 49/11 434) than that in other ethnic groups, and highest in the illiterates (1.92%, 63/3 285). ELISA showed that the positive rate for serum antibody against hydatid cyst was 3.60% (265/7 369) in 7 369 students examined, and showed a trend of decrease from 2009 (6.06%, 37/611) to 2014(2.59%, 22/850); the positive rate was higher in agricultural areas (3.99%, 207/5 193) than that in pastoral areas (2.89%, 3/1 487) and towns (2.18%, 15/689)(P<0.05), and highest in Han students (4.06%, 177/4 361). The hydatid cyst infection rate in 4 677 livestocks was 13.04% (610/4 677), decreased from 25.05% (127/507) in 2009 to 8.80% (44/500) in 2014. The positive rate of hydatid cyst antigen in dog feces was 5.18% (365/7 049), decreased from 16.98% (81/477) in 2009 to 2.56% (41/1 600) in 2014. Conclusion The morbidity of echinococcosis showed a trend of decrease in Wenquan County, and most of the patients are young adults. There remains a high level of echinococcosis morbidity, positive rate of serum echinococcosis antibody in 6-12-year-old students, infection rate in livestocks, and existence of hydatid cyst antigen in dog feces.

    Cloning and Ion Transportation of Cryptosporidium andersoni ATP-binding Cassette 1 Gene
    WANG Ju-hua1, WANG Pan1, LIU Chuan-huan1, SUN Xiu-mei1, XIE Qian-qian1, XUE Xiu-heng2 *
    2016, 34(4):  6-326-330. 
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    Objective  To investigate the transportation of intracellular and extracellular K+, Ca2+, Na+ and Mg2+ under the function of Cryptosporidium andersoni ATP-binding cassette(CaABC) 1 gene.  Methods  CaABC1 gene was amplified by PCR using specifically designed primers. The eukaryotic expression plasmid pEGFP-C1-CaABC1 was constructed, and transfected into mouse intestinal epithelial cells via liposome transfection. The blank(with no transfection) and control groups(transfected with empty plasmid pEGFP-C1) were also set. Changes in intracellular and extracellular K+, Ca2+, Na+ and Mg2+ concentrations were examined by the ion concentration assay kit. Results PCR amplification resulted in a 544 bp product. The recombinant plasmid pEGFP-C1-CaABC1 was successfully constructed. Green fluorescence was seen in the control and transfected groups, but not in the blank group. The concentrations of K+, Ca2+, Na+ and Mg2+ in intracellular fluid were (5.51 ± 0.51), (1.98 ± 0.06), (108.33 ± 1.33) and (0.93 ± 0.03) mmol/L in the blank group; (6.25 ± 0.70), (1.90 ± 0.13), (107.73 ± 1.79) and (0.87 ± 0.05) mmol/L in the control group; and (14.84 ± 0.90), (3.40 ± 0.14), (127.64 ± 1.49) and (1.72 ± 0.20) mmol/L in the transfected group. The concentrations of K+, Ca2+, Na+ and Mg2+ in extracellular fluid were (12.72 ± 0.83), (3.72 ± 0.03), (116.83 ± 1.04) and (2.02 ± 0.18) mmol/L in the blank group; (10.11 ± 0.90), (3.58 ± 0.06), (115.89 ± 1.86) and (1.71 ± 0.41) mmol/L in the control group; and (5.77 ± 0.21), (1.29 ± 0.18), (96.21 ± 1.19) and (0.64 ± 0.02) mmol/L in the transfected group. There were significant differences in K+, Ca2+ and Mg2+ concentrations between the transfected group and the control group.  Conclusion  CaABC1 participates in the transportation of K+, Ca2+ and Mg2+.

    Identification, Expression and Antigenicity Analysis of  Serpin B6 of Taenia solium
    LIU Guang-xue1, ZHANG Shao-hua1, GUO Ai-jiang1,2, HOU Jun-ling1, WEI Yan-ling1, WANG Shuai1, LUO Xue-nong1,2*
    2016, 34(4):  7-334-338,345. 
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    Objective To identify and express serpin B6 of Taenia soliumTsserpin B6) and explore its possible use as a diagnostic antigen. Methods Primers for Tsserpin B6 were designed according to T. solium genome and transcriptome data. The Tsserpin B6 gene was amplified from the total RNA of T. solium cysticercus and subsequently analyzed by bioinformatics. Multiple amino acid sequence alignments of Tsserpin B6 and other parasites serpins were created using the Clustal X1.83. Phylogenetic analyses were performed using the MEGA 6.0. The recombinant expression vector pET-30a-Tsserpin B6 was constructed and expressed in E. coli strain BL21 (DE3). The expressed proteins were purified, isolated by SDS-PAGE, and analyzed by Western blotting using pig serum infected with T. solium cysticerci. Results The complete reading frame of Tsserpin B6 was 1 131 bp and encoded a protein of 376 amino acids. The encoded protein had a conservative reactive center loop and distinctive domains of NEEGAE and FTVDHPFLF, and harbored 9 potential linear B cell epitopes. The expressed products of Tsserpin B6 mainly existed as an inclusion body, and reacted with pig serum infected with T. solium, resulting in a specific band at the Mr 53 000. Conclusion The Tsserpin B6 gene was successfully cloned, and its expressed products can be recognized by pig serum infected with T. solium.

    Polymorphism Analysis of Plasmodium falciparum K13 Gene #br# Kelch Domain Associated with Resistance to Artemisinin in #br# Yunnan Province
    SUN Ai-ming1,2, DONG Ying2*, CHEN Meng-ni2, XU Yan-chun2, DENG Yan2, MAO Xiang-hua2, WANG Jian2
    2016, 34(4):  8-339-345. 
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    Objective To investigate the polymorphism of Plasmodium falciparum K13 gene kelch domain region and provide basis for understanding the artemisinin resistance of falciparum malaria in Yunnan Province. Methods The filter blood samples and relative information of falciparum malaria cases were collected in 16 prefectures of Yunnan Province from January 2013 to December 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the China Information System for Disease Control and Prevention Epidemic Registration. The K13 gene kelch domain region was amplified by nested PCR, sequenced, and blasted against the reference strain 3D7(PF3D7_1343700). The K13 gene kelch domain region polymorphism was analyzed with Mega 5.04. The variable sites and genetic distance between sequences were analyzed. The constituent ratio of amino acid mutation sites was calculated and analyzed with χ2 test. Results A total of 202 blood samples were collected from 2013 to 2015, comprising 190 from imported cases, 12 from local cases in Yunnan Province. The constitutent ratio of infection cases were 30.7% (62/202), 34.2% (69/202) and 35.1% (71/202) respectively, increased year by year. The K13 gene kelch domain was successfully amplified from 192 samples and 190 were successfully sequenced, detecting missense mutation of K13 gene in 66 samples, the mutation rate was 34.7% (66/190). The detection rate of K13 gene mutation was 40.9% (27/66), 37.9% (25/66) and 21.2% (14/66) respectively, decreased year by year. In this study, ten types of mutations were detected, which were F446I, A578S, N458Y, P574L, A676D, G449A, C469Y, V494I, E556D and S16L. The highest mutation rate occurred in F446I which was 72.7% (48/66). The proportion of F446I mutation type was 58.3% (28/48) in an age-range of 18-56 years, 70.8% (34/48) in farmers, and 91.7% (44/48) in patients with infection source in Southeast Asia, all significantly higher than that of other groups with the same characteristics (41.7%, 20/48; 29.2%, 14/48; and 8.3%, 4/48, respectively)(χ2=4.633, 5.556 and 5.152, both P<0.05). There was a 248 bp homologous sequence in the 190 sequences, composed of 235 conservative sites (94.8%), 13 variable sites (5.2%), 5 parsim-info sites (2.0%), and 8 singleton sites (3.2%). The genetic distance among the 190 sequences ranged 0.000-0.036, with an average of 0.001±0.001. Conclusion There are 10 types of mutations in the K13 kelch domain in Yunnan Province, the predominant mutation type was F446I.

    The Impact of the Malaria National Strategy Application Program of the Global Fund on Malaria Elimination in Henan Province
    DENG Yan, CHEN Wei-qi, ZHANG Ya-lan, QIAN Dan, LIU Ying, ZHOU Rui-min, ZHANG Hong-wei*
    2016, 34(4):  9-346-349,354. 
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     Objective To analyze the impact of the national strategy application (NSA) program of the global fund on malaria elimination in Henan Province. Methods The internal control system on project, fund and supplies was developed to ensure the normalized implementation of NSA program in Henan Province. The multi-channel communication platform including telephone, e-mail and so on was established for data’s progressively reporting, review and summary. The training, supervision and assessment were implemented to ensure that the data was real and correct. The data derived from the NSA program and other project involved with malaria control in the Province from 2009 to 2012 was collected and compared. Changes on human resources, financial input, supplies purchasing and the main malaria control measures before and after the implementation of the program were analyzed. Results A total of 29.72 million yuan financial support which accounted for more than half of the total financial input on malaria control at the same time in the Province was provided by the NSA program. A total of 3 966 microscopists and 61 415 health workers from primary health care received skill training on malaria dectection and control. After implementation of the program,  there was an increase in the diagnosis rate by blood test from 2009 (45.67%, 738/1 616) to 2012 (99.36%, 155/156) (χ2=161.91. P<0.01). The malaria incidence showing a step-wise decline from 2009 to 2012 which were 1.71/100 000, 0.94/100 000, 0.34/100 000 and 0.17/100 000 respectively, with an increase in imported cases(χ2=1 310.70, P<0.01) and no report of indigenous malaria cases in 2012, for the first time. The reported cases in counties of the NSA program during 2009-2012 constituted 98.08%(1 585/1 616), 92.75% (831/896), 80.25% (252/314) and 67.31% (105/156) of all malaria cases reported in that year in the Province (χ2=300.38, P<0.01). The awareness rates on malaria prevention and treatment among residents and students were 94.65% (25 246/26 673) and 93.73% (16 328/17 421) in 2012, both significantly higher than the baseline in 2009 (63.88%, 5 755/9 009; 53.39%, 3 170/5 937) (χ2=5 594.01, P<0.01; χ2=5 221.53, P<0.01). Conclusion The implementation of the NSA program has motivated the elimination of malaria in Henan Province.

    Pathogen Identification for An Imported Case with#br# African Trypanosomiasis
    SUN Yi1, HUANG Wei-hua1, NIU Zi-guang1, WANG Hui1, GUO Jie1, HU Xiao-xia2, YU Jing-long1, ZHOU Dao-yin1, WENG Ya-li3, DENG An-mei1*
    2016, 34(4):  10-350-354. 
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    Objective To perform laboratory diagnosis for an imported case of human African trypanosomiasis and identify the pathogen. Methods Clinical and epidemiological information was collected. Blood and cerebrospinal fluid samples were collected, stained with Wright-Giemsa, and microscopically examined. Genomic DNA from the blood samples was amplified with primers specific for Trypanosoma sp. expression site-associated gene(ESAG), Trypanosoma brucei gambiense specific glycoprotein(TgsGP) and 18S rRNA(M18S-Ⅱ-Tb) gene, and Trypanosoma brucei rhodesiense specific serum resistance associated (SRA) gene. Complete blood count, blood chemistry, and CSF examination were also conducted. Results The patient had a 4-month history of lower extremity weakness and swelling of surface lymph nodes. Physical examination showed somnolence, and occasional emotional abnormalities, accompanied by anemia(hemoglobin 85 g/L), electrolyte disturbance(sodium 124 mmol/L; chlorine 87 mmol/L) and significantly increased nonspecific immune globulin protein (globulin 63 g/L). Epidemiological survey showed that the patient suffered insect bites and stings for several times during his work in the Republic of Gabon in Africa. Microscopic examination revealed flagella of trypanosome in peripheral blood. PCR amplification produced bands of 286, 308, and 150 bp with primers specific for ESAG, TgsGP and M18S-Ⅱ-Tb, respectively. Conclusion The patient was diagnosed with Trypanosoma brucei gambiense infection from the clinical information, epidemiological history, etiology and PCR results.

    Survey on Toxoplasma gondii Infection in Depressed Patients and Genotype Identification in Guizhou Province
    ZOU Cai-yan1, HE Jun2, YIN Ji2, BI Bin2, QIN Ying2, PENG Feng-tao1, XIA Yu1, TIAN Meng-yuan3, LI Yong-nian4 *
    2016, 34(4):  11-355-360. 
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    Objective To survey on Toxoplasma gondii infection in depressed patients in Guizhou Province and identify the genotype of T. gondii. Methods Enzyme-linked immunosorbent assay(ELISA) was applied to detect the T. gondii-specific antibodies IgG, IgM and circulating antigens (CAg) of T. gondii in 141 patients and 150 healthy subjects. The specific repeated DNA fragment(529 bp) of T. gondii was amplified by PCR. The genotype of T. gondii was determined by multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RFLP). Results ELISA showed that the positive rate of anti-T. gondii antibody in depressed patients and healthy subjects was 21.3%(30/141) and 7.3%(11/150), respectively. The positive rate of IgG in depressed patients was 18.4% (26/141), significantly higher than that in healthy subjects (7.3%, 11/150)(P<0.05). The positive rate of IgM and CAg in depressed patients was both 1.4% (2/141), while these were not found in healthy subjects. PCR revealed one patient positive for T. gondii, whose genotype was further identified to be Toxo DB #9(Chinese 1 type) by Mn-PCR-RFLP. Conclusion The positive rate of T. gondii is higher in depressed patients than in the healthy subjects in Guizhou Province. The genotype of T. gondii detected in one depressed patient is the Chinese 1 type.

    Epidemiological Survey on Human Intestinal Protozoa in#br# Xinjiang Uygur Autonomous Region in 2015
    Mamatjan UMAR1,2, CHEN Xiao-ying3, Yisilayin OSMAN1, Adili SIMAYI1, HOU Yan-yan1, Yalikun MAIMAITIYIMING1, XIAO Ning2*
    2016, 34(4):  12-361-365. 
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    Objective An epidemiological survey was made on human intestinal protozoa in Xinjiang Uygur Autonomous Region(Xinjiang) to evaluate recent control achievements and provide basis for making specific control strategies. Methods Regions in Xinjiang were categorized by types of ecological system and geographical characteristics into five ecological areas(types I-V) according to the National Ecological Function Stratification issued by the Ministry of Environment Protection and Chinese Academy of Science and Technology. Stratification sampling was made in each ecological area. Feces from human of all ages were collected for morphological identification of protozoan species using the Iodine Liquid Direct Smear Method. Results In the five ecological areas, 26 886 people from 132 survey sites in 39 counties(cities) were examined, with an examination rate of 81.47% (26 886/33 000). The infection rate was 0.32%(85/26 886). Four species of intestinal protozoa were detected, i.e., Entamoeba histolytica, Giardia lamblia, Blastocystis hominis, and Entamoeba coli, with an infection rate of 0.22% (60/26 886), 0.03% (9/26 886), 0.01% (2/26 886) and 0.61% (17/26 886), respectively. Of the five areas, the type IV area had the highest infection rate of 0.75%(28/3 758)(P<0.05). Besides, the infection rate was higher in males (0.24%, 33/13 623) than that in females (0.39%, 52/13 263) (P<0.05), higher in age ranges of 21-30(1.40%, 16/3 959) and 31-40 years(0.46%, 22/4 799) than other age groups (P>0.05), higher in housewives (0.48%, 2/418) than those with other occupations, higher in the Hui group (0.61%, 15/2 445) than that in other ethnic groups, as well as highest in populations with a primary education level (0.37%, 35/9 375) and lowest in those with a college level or above(0.20%, 8/3 945). Conclusion The human intestinal protozoa infection was at a low level in Xinjiang in 2015.

    Application of Confocal Laser Scanning Microscopy in Detection of Demodex Mites
    XIAO Jia, GUO Ai-yuan, HUANG Jian, CHEN Jing*
    2016, 34(4):  13-366-369. 
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    Objective To explore the application value of confocal laser scanning microscopy(CLSM) for detection of facial Demodex mites. Methods One hundred and twenty patients without basic diseases visiting the Department of Dermatology of the Third Xiangya Hospital for papules, pustules, erythema, and scales and suspected to have facial demodicosis were selected. Basic information and types of facial lesion were recorded. CLSM was performed to examine Demodex infection. Twenty patients with positive infection as indicated by CLSM were randomly selected for further verification with microscopy. Results Among the 120 patients, 59 were positive for facial demodicosis as indicated by CLSM, with a detection rate of 49.2%, 61.1% of males and 39.4% of females. The 20 patients with further microscopic examination were all diagnosed as Demodex folliculorum infection. Among the 120 patients, the Demodex detection rate showed a trend of increase with age, with the highest rate (72.7%, 16/22) in the group of 40-49 years[P<0.05 versus the 21.1% (4/19) in the group of 10-19 years and the 37.8% (14/37) in 20-29]. In addition, there was a significant difference in the percentage of patients with <5 mites and ≥5 mites in area of 4 mm×4 mm between type I (mainly erythema and scales)(80.6%, 29/36; 19.4%, 7/36, respectively; P<0.01) and type II lesions (mainly papules and pustules) (52.2%, 12/23; 47.8%, 11/23, respectively; P>0.05)(P<0.05). Conclusion CLSM is an efficient technique for detecting facial Demodex, and is advantageous as it offers in-situ, noninvasive, painless, real-time and dynamic detection.

    Hydatidosis Prevalence in 6- to 12-year-old Children#br# in Southern Xinjiang
    ZHAO Jiang-shan,HOU Yan-yan*,ZHANG Hai-ting,Ruziguli-Zhumahong
    2016, 34(4):  14-370-372,376. 
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    Objective To understand the hydatidosis prevalence in 6- to 12-year-old children in southern Xinjiang Uygur Autonomous Region and provide scientific basis for formulation of control measures. Methods One primary school was selected from each of the pastoral area, pastoral-agricultural area, agricultural area, and township area in 42 counties (cities) of southern Xinjiang, using the stratified sampling method. With the consent of their parents, B ultrasonic abdominal scan and venous blood collection were performed on all the children (6-12 years) in the selected schools. Serum level of echinococcus antibody IgG was determined by ELISA. Hydatidosis prevalence and the serum level of IgG in these children were analyzed. Results A total of 80 429 children were examined from 168 primary schools. B ultrasonic scan revealed hydatidosis in 9 children (0.01%), comprising 3 cases in Kezhou, 2 in Aksu, 2 in Bazhou, and 2 in Kashi. ELISA results showed that 4 189 children were positive for serum IgG, with a positive rate of 5.21%. The positive rate was highest in Kashi (8.41%, 2 143/25 495), followed by Aksu (5.69%, 913/16 051), with significant difference between different areas (χ2=977.303, P<0.01). The positive rate was lowest in 6-year children (2.13%, 44/2 065) and highest in 11-year children (5.68%, 822/14 462) (χ2=48.221, P<0.01). In addition, the positive rate was highest in Uighur children(5.19%, 3 899/75 115), followed by Mongolian children (4.27%, 68/1 592), with no significant difference between ethnic groups (χ2=4.072, P>0.05). Among the children of residents, children with a nomadic lifestyle, and children settled in winter and living in a nomadic lifestyle in summer, hydatidosis occurred in 7 residents and 2 children who settled in winter and living in a nomadic lifestyle in summer; and the positive rate of serum antibody was 5.45% (184/77 512), 2.97% (3/101), 0.07% (2/2 816), respectively(χ2=148.609, P<0.01). Conclusion There is a low hydatidosis prevalence in 6- to 12-year-old children in southern Xinjiang, but the positive rate of serum echinococcus IgG in kashgar region, Uighur ethnicity, and children of the resident group are relatively high.

    Gaps in Clonorchiasis Control in China
    QIAN Men-bao, CHEN Ying-dan, ZHOU Xiao-nong
    2016, 34(4):  15-373-376. 
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    Clonorchiasis is currently the most important food-borne parasitic disease in China. In this review, gaps in the control of clonorchiasis in China are explored through problem tree analysis. From the perspective of disease control, clonorchiasis hyper-endemicity is associated with the implicit disease burden, incomplete epidemiological map, unclear epidemiological determinants, and a lack of surveillance and report system. Thus, studies are needed to overcome these problems, to promote clonorchiasis control and elimination in China.

    Application of DNA Microarray Technology in Human Trypanosomiasis Research
    CHEN Mu-xin, CHEN Jia-xu*
    2016, 34(4):  16-377-381. 
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    With the rapid development of molecular biological techniques, DNA microarray has shown great advantage in biology and medicine as it allows high-throughput measurement of various biological parameters. Trypanosomiasis remains the focus among numerous human blood parasitic diseases. The DNA microarray is a useful technique for studying the trypanosome genome and parasite-host interaction, as well as for vaccine screening and drug development. This review gives an update on the application of DNA microarray in human research on Trypanosoma brucei and Trypanosoma cruzi.

    Immunomodulatory Effect of Trichinella spiralis and its Derivative#br# Products in Allergy and Autoimmune Diseases
    DING Yi-han,WANG Xiao-li,SONG Di,WU Qi,YANG Xiao-di*
    2016, 34(4):  17-382-386. 
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    Trichinella spiralis and its derivative antigens stimulate T cell activation by inducing dendritic cell semimaturation, thereby regulating the Th1/Th2 type immune response and alleviating or inhibiting allergy and autoimmune diseases. The regulatory T cell and anti-inflammatory factors play important roles in this immunomodulatory process. This article reviews the modulatory effects and their mechanisms of Trichinella spiralis and its derivatives in autoimmune disorders.

    Interpretation of Criteria for Diagnosis of Toxoplasmosis
    YAN Xiao-lan1, WEN Li-yong1*, GUAN Ya-yi2, ZHANG Jian-feng1, LIN Dan-dan3
    2016, 34(4):  18-387-388,封三. 
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    The criteria for Diagnosis of Toxoplasmosis(WS/T 486-2015)(referred to as the Criteria) was compiled following the Management Measures for Health Criteria and GBT 1.1-2009 Standardization Working Guidelines. The Criteria is composed of six chapters, including the range of application, terms and definitions, diagnostic principle, diagnostic standard, and differential diagnosis. Four informative appendices(etiology, epidemiology, clinical manifestation, and differential diagnosis) and one normative appendix(laboratory examination) are appended. The Criteria was issued by the National Health and Family Planning Commission of the People’s Republic of China through No.21 of Chinese Health Announcement in 2015. The Criteria provides for the first time technical reference for diagnosis of toxoplasmosis in medical institutions and disease control institutions.

    Exploration on the Technology of Counter Staining of Fluke
    XIE Wen-ting1, SHUAI Mu-lan1, WANG Shao-sheng2, ZHAO Jin-hong2*, LI Chao-pin2
    2016, 34(4):  19-323-325. 
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     Fixed samples of Clonorchis sinensis and Fasciolopsis buski were stained with acetocarmine and malachite green, or stained with acetocarmine only. The samples displayed three different colors after staining with acetocarmine and malachite green. The digestive system, excretory system and the surrounding muscle tissue were stained reddish, the uterus was bright green, and the vitellarium at each side of the worm was tan. Staining with the two dyes resulted in clear structure and moderate degree of staining, and allowed three-dimensional observation, while staining with acetocarmine highlighted the testis tissue. Therefore, combination of the two staining methods is recommended in teaching and research to more effectively facilitate observation.

    Efficacy of Capture and Ligation Probe-PCR with#br# Pooling Strategy in Detection of Plasmodium spp.
    ZHOU Yao-wu1, LIN Zu-rui1, CHENG Zhi-bin2, ZHENG Zhi2,ZHOU Xing-wu1, ZHOU Zi-you1, WANG Duo-quan3, LI Jian-xiong1, ZENG Xu-can1, SUN Xiao-dong1*
    2016, 34(4):  20-330-333. 
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    The capture and ligation probe-PCR(CLIP-PCR) with pooling strategy method and microscopy were applied on 100 clinical samples(7 positive and 93 negative samples) from the malaria reference laboratory in Yunnan Province. By calculating the detection rate, sensitivity, specificity, detection time and detection cost, the efficacy of the CLIP-PCR with pooling strategy method in detecting Plasmodium spp. was evaluated. The CLIP-PCR with matrix pooling strategy successfully detected Plasmodium spp. in all the 7 positive samples. Its sensitivity and specificity relative to the microscopy as a gold standard were both 100%. The detection time for all the samples by CLIP-PCR was 5.0 h, 85.0% shorter than that by microscopy(33.3 h), and the detection cost was 300 yuan, 75.0% less than that by microscopy (1 000 yuan).