Table of Content

30 December 2017, Volume 35 Issue 6

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ORGINAL ARTICLE

Malaria situation in the People’s Republic of China in 2016

Li ZHANG, Jun FENG, Shao-sen ZHANG, Shan JIANG, Zhi-gui XIA, Shui-sen ZHOU

2017, 35(6):  515-519. 

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Objective To analyze epidemiological characteristics of malaria in China in 2016 so as to provide evidence-based proof for target interventions on malaria elimination. Methods The data of Malaria Annual Reporting System in 2016 were collected. And the epidemic situation, regional distribution, species composition, diagnosis type and infection source were analyzed by Microsoft Excel 2010 software. Results Totally 3 321 malaria cases were reported in 687 counties of 30 Provinces/Municipalities/Autonomous Regions (P/M/As) in 2016, which was increased by 1.0% compared with that in 2015(3 288). The cases were reported primarily from Provinces of Yunnan (12.4%, 413/3 321), Sichuan (9.8%, 327/3 321), Jiangsu (9.3%, 308/3 321), Guangxi (9.2%, 305/3 321) and Shandong (7.7%, 256/3 321). Of all the cases, 3(0.1%, 3/3 321) P. vivax cases were indigenous, from Yingjiang county of Yunnan (2) and Chayu county of Tibet (1). Meanwhile, 3 317 (99.9%, 3 317/3 321) cases distributed in the 30 P/M/As. A transfusion-transmitted P. falciparum case was reported in Jiangsu Province. In addition, 15 (0.5%, 15/3 321) cases were diagnosed clinically, and 3 306 (99.5%, 3 306/3 321) of the reported cases were confirmed in reference laboratories, comprising 712 P. vivax cases(21.5%, 712/3 306) , 2 158 P. falciparum cases (65.3%, 2 158/3 306), 64 P. malariae cases (1.9%, 64/3 306), 311 P. ovale cases (9.4%, 311/3 306) and 61 mixed-infection cases(1.8%, 61/3 306). Furthermore, 185 cases (5.6%, 185/3 321) with severe clinical symptoms were reported in 14 P/M/As, with 15 deaths (0.5%, 15/3 321) in 8 P/M/As. Conclusion The local transmission of malaria has been effectively controlled, though malaria elimination in the border areas of Yunnan Province and Linzhi Prefecture of Tibet Autonomous Region are still challenging. It is needed to strengthen the monitoring and management of imported cases, and to make risk assessment for possible malaria re-transmission.

An overview on cooperation strength between China and the Global Fund to Fight AIDS, Tuberculosis and Malaria in developing multilateral malaria control projects

Zhi-gui XIA, Ming XU, Jin-kou ZHAO, Sheng-jie LAI, Xiao-nong ZHOU

2017, 35(6):  520-526. 

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Objective To strengthen cooperation between China and the Global Fund to Fight AIDS, Tuberculosis and Malaria in developing multilateral malaria control projects. Methods Data of China malaria cases imported from other countries were extracted from recent literatures. The 2017-2019 funding cycle data of the Global Fund were downloaded from its website. Descriptive statistical analysis was performed using IBM SPSS Statistics 22, and geographical mapping was conducted using QGIS 2.18.Results In 2011-2014, the imported malaria cases of China were from 63 countries, of which 38 countries had an average annual export of ≤ 20 cases, 11 countries had 21-40 cases, 9 countries had 41-100 cases and 5 countries （Myanmar, Ghana, Angola, Equatorial Guinea and Nigeria） had > 100 cases. In the 2017-2019 funding cycle, the Global Fund allocated USD 3.3 billion (exclusive of the catalytic investment) in the malaria field. Among the 67 eligible countries/regions, the main investments went to High-Impact Africa (funds accounting for 55.7%), Western Africa (12.5%), Central Africa (7.8%) and High-Impact Asia (9.9%); the numbers of countries/regions accounting for < 1.0%, 1.0%-1.9%, 2.0%-4.5%, 5.0%-6.0% and > 9.0% of total funds were 36, 16, 11, 2 and 2, respectively. As to the application approach, 37 were through program continuation, 15 through full review and 15 through tailored review. Fifty applications have entered the grant-making stage. Fifty-two (accounting for 85.3% of China’s imported malaria cases in 2011-2014) out of 63 countries that exported malaria to China are eligible for 87.4% of the total malaria fund.Conclusion It is time to start multilateral cooperation between China, the Global Fund, and some selected countries to jointly promote global malaria control and elimination and for personal security against malaria when being out of China, management of imported malaria, and fulfillment of the Belt and Road Initiative of China.

Identification and expression of the Aedes aegypti serpin gene family

Jin-zhi CHENG, Jian LIU, Hui ZHAI, Qing-qiao LV, Feng YAN, Zheng-ling SHANG, Jia-hong WU

2017, 35(6):  527-535. 

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Objective To identify serpin genes from the whole genome of Aedes aegypti, and analyze the structure of the gene and its expression at various developmental stages and in different tissues. Methods The serpin gene family was identified in Ae. aegypti genome database. Sequences were aligned for homology analysis and conservation analysis using Vector NTI11.0 softwares. The phylogenetic tree was constructed using MEGA7.0 software. qRT-PCR was performed to examine serpin expression at different developmental stages (eggs, larva of Ⅰ-Ⅳ stages, pupa, male mosquito and blood-unfed female mosquito) and in different tissues(salivary gland, midgut, fat body and ovary) of blood-unfed female mosquitos. Results Twenty-six gene sequences of serpin were identified from Ae. aegypti genome. Bioinformatics analysis revealed that 19 gene sequences harbored a signal peptide, and 16 were predicted to be inhibitory on enzymes. Phylogenetic analysis demonstrated that the serpin gene family of Ae. aegypti was divided into A, B and C clusters, in which cluster B was split into 4 groups. Each cluster showed homology, and members of each cluster were conservative. Results of qRT-PCR showed that AaSRPN25 was expressed specifically in female mosquitos (0.074 ± 0.015, P < 0.01), while AaSRPN19, AaSRPN20, AaSRPN21 and AaSRPN22 were enriched in male mosquitos. AaSRPN23 had a high expression level not only in the stage-Ⅰlarvae(22.9 ± 5.28) but also in the salivary gland (4.24 ± 1.39)(P < 0.01); AaSRPN25 were highly expressed specifically in the salivary gland (74.44 ± 25.67, P < 0.01); AaSRPN6 and AaSRPN14 had the highest expression in the mid gut(1.80 ± 0.33 and 0.703 ± 0.501, respectively); AaSRPN17 and AaSRPN20 had the highest expression in the fat body (0.34 ± 0.09 and 0.15 ± 0.03, respectively). Conclusion 26 Serpin sequences of Ae. aegypti have been identified in the whole genome, in which 19 sequences owned the signal peptide, and 16 sequences were speculated to have an inhibiting enzyme effect.

Analysis of knowdown resistance gene mutation in Culex tritaeniorhynchus resistant to DDT and deltamethrin in Yunnan Province, China

Jin-yong JIANG, Hui-ying CHEN, Hong-ning ZHOU, Ya-jun MA

2017, 35(6):  536-540. 

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Objective To detect the knockdown resistance（kdr） gene mutations in Culex tritaeniorhynchus resistant or susceptible to DDT and deltamethrin, in order to elucidate the relationship between the resistant phenotype and kdr mutations.Methods C.tritaeniorhynchus mosquitoes were captured in Zhaoyang, Mang Shi, Jiang Cheng and Meng Lian in Yunnan Province, and exposed to DDT and deltamethrin, respectively.The resulting alive and dead ones were counted.Genomic DNA was extracted by individual mosquitoes.The kdr gene was amplified by PCR and analyzed.The frequency of kdr genotypes was estimated in resistant and susceptible samples.The relationship between kdr genotypes and resistant phenotypes was analyzed by Chi-square test.Results A total of 411 mosquitoes were genotyped for the kdr gene.Mutation was detected at codon 1014, yielding two alleles, the wild-type TTA/L and the mutant TTT/F, and three genotypes, the wild-type homozygote L/L （frequency, 96.35%; 396/411）, the mutant homozygote F/F （0.73%; 3/411） and the wild/mutant heterozygous L/F （2.92%; 12/411）.The frequencies of mutant genotype in DDT-resistant and -susceptible individuals were 3.42% （4/117） and 3.88% (4/103), respectively; while those in deltamethrin-resistant and -susceptible individuals were 3.96% (4/101) and 3.33% （3/90）, respectively.Chi-square test revealed that there was no correlation between kdr genotype frequency and the resistance phenotypes to either DDT （χ² = 0.034, P > 0.05） or deltamethrin （χ² = 0.053, P > 0.05）.Conclusion We found a novel L1014F mutant allele for kdr, albeit with a low frequency.There is no correlation between kdr gene mutation and the resistance phenotypes to either DDT or deltamethrin.

Endemic situation at schistosomiasis surveillance sites in China in 2016

Jia-ning JIN, Hui DANG, Li-juan ZHANG, Ying-jun QIAN, Shan LV, Shi-zhu LI, Xiao-nong ZHOU, Jun-ling SUN, Jing XU

2017, 35(6):  542-548. 

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Objective To analyze the endemic situation at the schistosomiasis surveillance sites in China in 2016, in order to provide scientific basis for evaluation of schistosomiasis control effect.Methods According to the National Schistosomiasis Surveillance Programme （version 2014）, 454 national surveillance sites were selected in counties (cities or districts) with schistosomiasis prevalence and potential counties （cities or districts） with prevalence.Infections in residents, floating populations, domestic animals and Oncomelania snails were monitored and analyzed in four types of endemic counties.Results A total of 129 971 residents received indirect hemagglutination（IHA） test in 2016, of whom 3 852 showed positive results.Of them 3 801 received etiological test, 21(19 found in Hunan Province and 2 found in Jiangxi Province) showed infections in fecal samples, with an infection rate of 0.02% (21/129 971).Serological examination was also performed in 97 474 persons of the floating population, and 980 showed positive results.Of them 953 received etiological test, 9 (8 found in Zhejiang Province and 1 found in Hunan Province, all were imported cases) showed infections in fecal samples, with an infection rate of 0.01%（9/97 474）.No acute schistosomiasis was reported in any surveillance site.In addition, 12 769 cattle received examinations and only 1 was positive for infection, with a cattle infection rate of 0.007 8%.The snail survey covered an area of 22 371.69 hm² (1 hm² = 10 000 m²) and snails were found in an area of 6 999.57 hm², including a newly detected area of 136.77 hm² distributed in Shanghai and Anhui Province.Snail re-emergence was detected in a total area of 125.22 hm² in provinces other than Hunan, Hubei and Guangdong Provinces, but no schistosome infection was found in them.Conclusions There was a stable endemic situation in China in 2016.However, in some provinces snails still appeared for the first time or re-emergence was detected.

Expression and evaluation of diagnostic candidate antigens from Babesia microti

Xiu-feng LIU, Jia-hui SUN, Bin XU, Jun-hu CHEN, Wei HU

2017, 35(6):  549-553. 

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Objective To clone and express Babesia microti diagnostic candidate antigens, and to preliminarily evaluate their diagnostic value.Methods Immune screening was performed using bioinformatics method on the cDNA library of Babesia microti constructed previously in our lab, resulting in 5 positive clones, namely Bm2, Bm4, Bm6, Bm9 and Bm15.The open reading frames（ORFs）of their amino acid sequences were predicted.The desired genes were amplified using the pBluscript plasmids of positive clones.The products were cloned into pET28a and transformed into E.coli BL21(DE3) system.Expression was induced with isopropyl-β-D-thiogalactopyranoside（IPTG） at a final concentration of 1 mmol/L.Expression and solubility of the recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The insoluble recombinant proteins were purified using Micro Protein PAGE Recovery Kit.The sensitivity and specificity of recombinant antigens were evaluated with ELISA method using mouse sera infected with B.microti and their cross-reactivity with sera of malaria patients was evaluated.Results The five candidate antigen genes were amplified by PCR and resulted in protein bands of 480, 300, 750, 200 and 700 bp, respectively, as predicted by bioinformatics software.The sequences were verified to be consistent with cDNA library of B.microti.The Bm4, Bm6 and Bm15 genes were induced by IPTG to express as inclusion bodies with relative molecular mass of 13 000, 30 000 and 30 000, respectively.However, expression of Bm2 and Bm9 was not achieved.Bm4, Bm6 and Bm15 proteins were extracted with Micro Protein PAGE Recovery Kit and analyzed by SDS-PAGE, resulting in single protein bands.The sensitivity of Bm4, Bm6, Bm15 and BmSA1(control) to mouse sera was 15.0%, 55.0%, 80.0%, and 100.0%, respectively, while the specificity was 100.0%, 100.0%, 90.0%, and 100.0%, respectively.Bm4, Bm6 and Bm15 showed no cross-reactivity with sera of malaria patient, whereas BmSA1 showed some positive cross-reactivity with a 13.3% false positive rate.Conclusion Three diagnostic candidate antigens of B.microti are expressed and purified, and Bm15 shows promising sensitivity and specificity as B.microti diagnostics.

Prediction and identification of B cell epitopes of lactate dehydrogenase of Echinococcus granulosus

Shuai YAN, Ping YAN, Xiao-jin MO, Bin XU, Ting ZHANG, Wei HU, Ruo-ting ZHAN, Ping YE

2017, 35(6):  554-558. 

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Objective To predict and identify B cell epitopes of lactate dehydrogenase of Echinococcus granulosus(EgLDH), in order to advance the development of highly specific and sensitive diagnostic method for echinococcosis.Methods ESPript 3.0 online program and IEDB software was used to predict secondary structure, β-fold, Emini Surface Accessibility, Karplus & Schulz Flexibility, Kolaskar & Tongaonkar Antigenicity, Parker Hydrophilicity and Bepipred Linear Epitope of EgLDH.Based on the prediction, potential epitopes were synthesized in vitro to assess their diagnostic efficacy using ELISA.The sensitivity, specificity and cross-reactivity were evaluated using sera from patients with echinococcosis, cysticercosis and healthy participants.Cystic fluid antigen was the control.Results The predicted secondary structure of EgLDH was composed of 37.16% α-helix, 9.67% β-fold and 53.17% random coil, with four transmembrane domains.Seven potential B cell epitopes were predicted, of which four were synthesized, i.e.peptides PP-1（215-228）, PP-2 （189-200）, PP-4 （79-89） and PP-7 （21-33）, with a sensitivity of 52.9%（18/34）, 61.8%（21/34）, 82.3%（28/34） and 50.0%（17/34）, respectively, to sera from patients with echinococcosis.There were significant differences between PP-4 and PP-1, PP-2 as well as PP-7（χ² = 8.100, 5.143, 9.091, P < 0.05）.The sensitivity of cystic fluid antigen was 91.1% （31/34）, which was significantly different from those of PP-1, PP-2 and PP-7 （χ² = 9.600, 5.780, 10.530, P < 0.05）, but not PP-4 （χ² = 0.444, P > 0.05）.The specificity to normal serum was 100% （21/21）, 100% （21/21）, 95% （20/21） and 95% （20/21）, respectively, all comparable to that of cystic fluid antigen （95%, 20/21）.The cross-reactivity to cysticercosis was 0, 1/10, 2/10 and 2/10, respectively, while that of the cystic fluid antigen was 5/10.Conclusion The PP-4 epitope of EgLDH B-cell shows a high sensitivity and specificity to sera from patients with echinococcosis and may be a potential diagnostic antigen for hydatid disease.

Cloning, expression, and immunoreactivity analysis of antigen Eg-08002 from protoscoleces of Echinococcus granulosus

Hui YANG, Yin-qi ZHAO, Zi-hua LI, Jia-qing ZHAO, Hao WANG, Ya-na WANG, Ming-xing ZHU, Wei ZHAO

2017, 35(6):  559-562. 

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Objective To clone, express and analyze the immuno-reactivity of Eg-08002, an antigen screened out from protoscolex-stage Echinococcus granulosus.Methods Based on a previous study of transcriptome sequencing at the protoscolex phase and oncosphere, the protoscolex stage specific antigen Eg-08002 was identified and amplified by PCR and cloned into vector pET28a to construct recombinant plasmid pET28a-Eg-08002, which was then transformed into Escherichia coli JM109 for expression under the induction of IPTG.The recombinant rEg-08002 protein was purified with His-tag Ni-superflow affinity chromatography, and verified by rapid non-staining polyacrylamide electrophoresis.Immunoreactivity of rEg-08002 was analyzed by Western blotting, which was then assessed with sera from 12 patients with cysitic echinococcosis and 12 healthy individuals by ELISA.Results High expression of Eg-08002 was detected in protoscolex, with a length of 612 bp.The recombinant pET28a-Eg-08002 was constructed.Results of rapid polyacrylamide gel electrophoresis and Western blotting indicated that the recombinant protein rEg-08002 was highly expressed in E.coli JM109 in a predominant form of inclusion bodies, with an M_r of 23000, and was recognized by sera from mice with cystic echinococcosis.ELISA results showed that the average A₄₅₀ values with sera from the patients and healthy individuals were 1.245 ± 0.265 and 0.469 ± 0.006, respectively, with statistical difference（P < 0.05）.Conclusion The antigen Eg-08002 was identified with high expression in the protoscolex phase with a promising immunoreactivity as revealed by Western blotting and ELISA

Immunogenecity of recombinant Leishmania donovani Peroxidoxin-1, tryparedoxin peroxidase, hypothetical protein CAJ07026.1 and GDP-mannose pyrophosphorylase in BALB/c mice

Bao-qian JING, Yong-en XIE, Wei-min HU, Jian YANG, Chao-li WANG, Li FENG

2017, 35(6):  563-569. 

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Objective To evaluate the immune responses to recombinant Leishmania donovani peroxidoxin-1(Pxn1), tryparedoxin peroxidases(TryP), hypothetical protein CAJ07026.1(Hyp07026) and GDP-mannose pyrophosphorylase(GDPMP) in BALB/c mice.Methods The genes of L.donovani Pxn1, TryP, Hyp07026 and GDPMP were amplified by RT-PCR separately, and cloned into plasmid pQE30 to construct recombinant pQE30-Pxn1, pQE30-TryP, pQE30-Hyp07026 and pQE30-GDPMP, respectively.They were then transfected into Escherichia coli for expression under IPTG induction.rPxn1 and rTryP were purified with the NTA-Ni purification system under native condition, while rHyp07026 and rGDPMP were purified with the same system under denaturing condition.Then the recombinant proteins were used to immunize BALB/c mice (n = 5 for each) for 4 times and PBS was used as a control (n = 5).After that, RNA was extracted from spleen cells, and the transcriptional profiling of cytokines was examined by qRT-PCR microarray.Results PCR amplification of Pxn1, TryP, Hyp07026 and GDPMP produced fragments of 570, 610, 1 040 and 1 200 bp, respectively, sequences of which were consistent with those in GenBank as confirmed by the sequencing results.All of them were highly expressed in E.coli under IPTG induction, with molecular weights of 22 000, 23 000, 38 000 and 45 000 for rPXNT1, rTXNT, rHyp07026 and rGDPMP, respectively.Among the mRNAs of 84 cytokine genes expressed in the spleen cells of the BALB/c mice immunized with rGDPMP, rPXNT1, rTXNT and rHyp07026, significantly higher mRNA levels of IL-1f6( 96.22, 271.54, 108.51 and 250.15, respectively ), IL-17f (105.54, 35.71, 53.38 and 53.57, respectively ) and IL-10 (21.68, 27.51, 24.45 and 2.91, respectively ) were found in comparison to PBS control, with significantly lower mRNA levels of IL-4 (5.74, 2.43, 1.59 and 0.76, respectively), IL-12β (0.05, 0.26, 0.10 and 0.50, respectively), IL-18 (0.12, 0.21, 0.13 and 0.04, respectively ) and IFN-γ(3.59, 0.39, 0.29 and 0.63, respectively).Conclusion The findings suggest that the rTryP, rPxn1, rHyp07026 and rGDPMP of L.donovani can stimulate inflammatory responses and induce immune suppression.

Toxoplasma gondii ROP16 causes apoptosis of mouse brain nerve cells

De-jun GAO, Shuang CHANG, Xiu-mei SHAN, Wei-wei FAN, Zuo-hua MAO

2017, 35(6):  570-574. 

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Objective To construct a recombinant lentivirus expression vector containing Flag-tagged sequence of Toxoplasma gondii ROP16, and test its expression in nerve cells and its effect on apoptosis of brain nerve cells in mice.Methods The Flag-tagged ROP16 gene was amplified by PCR and cloned into lentiviral vector to construct the recombinant plasmid pCDH-CMV-copGFP-ROP16-Flag, which was then transformed into E.coli DH5α competent cells.The recombinant plasmids were verified with PCR, double enzymetic digestion and sequencing.Then they were packaged into virus to infect 293T cells.The green fluorescence was observed under a fluorescence microscope and the virus titer was calculated.The packaged lentivirus was then used to infect SH-SY5Y cells, accompanied by a pCDH-CMV-copGFP empty plasmid group.Expression of ROP16 was examined by Western blotting.The concentrated lentivirus was injected into the mouse prefrontal cortex M1 region by stereotactic injection.Two weeks later, the mice were sacrificed and frozen slices of the brain were prepared.Apoptosis of brain nerve cells was observed under a fluorescence microscope after TUNEL staining.Results As expected, PCR and double enzymetic digestion both resulted in a band of ~2 200 bp from the recombinant plasmid pCDH-copGFP-ROP16-Flag.Sequencing result was consistent with the ROP16 gene of Toxoplsma gondii RH strain (GenBank Accession No.GQ249093.1).Green fluorescence was seen under the fluorescence microscope with a titer of about 2E + 8 TU/ml.Western blotting analysis showed a specific band at a relative molecular weight of 120 000, suggesting that the recombinant plasmid could express ROP16 protein in SH-SY5Y cells.TUNEL staining revealed significantly more apoptotic cells in the ROP16 group than other groups.Conclusion The lentiviral vector pCDH-copGFP-ROP16-Flag expresses ROP16 protein in nerve cells and induces apoptosis of mouse brain nerve cells.

Fusion expression and identification of B cell epitopes of Toxoplasma gondii surface antigens（SAG）1 and 2

Zhao-zhe WANG, Rui XU, Yang HONG, Jiao-jiao LIN, Ke LU, Hao LI, Zhao-guo CHEN, Yao-jun SHI, Si-min WU, Jia-xin JIANG, Jia-jing LI, Chuan-gang ZHU

2017, 35(6):  575-579. 

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Objective To construct the prokaryotic expression system for B cell epitopes of Toxoplasma gondii surface antigens（SAG）1 and 2, and analyze their immunoreactivity.Methods The B cell epitope sequences of Toxoplasma gondii SAG1 and SAG2 were designed and synthesized according to the gene sequences of SAG1 and SAG2 published in GenBank.The target gene was subcloned into pET-28a(+), and expressed in Escherichia coli BL21 (DE3).Complete single colonies were then selected from LB medium containing kanamycin.After culture under shaking to an absorbance(A₄₅₀) value of 0.6-0.8, IPTG was added at a final concentration of 1 mmol/L to induce expression of the target gene.To obtain high concentrations of recombinant SAG(rSAG), the rSAG protein was purified using a His fusion protein purfication kit, and the resultant His-tagged purified protein was identified by Western blotting and ELISA.Results of PCR and double-enzyme digestion showed a specific protein band at ~909 bp.As expected, sequencing of the band further confirmed the construction of rSAG.SDS-PAGE revealed the rSAG molecular weight(M_r) of ~50 000.Western blotting showed specific binding of rSAG with a 1 ∶ 100 dilution of T.gondii-positive mouse serum.ELISA results showed a most positive reaction with the dilution of 1 ∶ 1 for recombinant protein and the dilution of 1 ∶ 50 for positive serum.Moreover, reactions between different dilutions of rSAG and different concentrations of T.gondii-positive mouse serum, and a good performance in distinguishing negative from positive serum.Conclusion The constructed recombinant protein rSAG is soluble and has good immunoreactivity.

Status of Cysticercus cellulosae infection and its impact on mathematical learning ability among school-aged children in Tibetan agricultural areas of Sichuan Province

Rui-xue YE, Yu-ju WU, Qing-zhi WANG, Min CAO, Tiao-ying LI, Xing-wang CHEN, Huan ZHOU

2017, 35(6):  580-585. 

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Objective To understand the status of Cysticercus cellulosae infection and its impact on the mathematical learning ability among school-aged children in Tibetan agricultural areas of Sichuan Province.Methods In October 2015, a multistage stratified cluster sampling method was used to employ five- and six-grade children in all primary schools located in agricultural areas in three Tibetan counties (Muli County in Liangshan Prefecture, Yajiang County in Ganzi Prefecture, and Ruoergai County in Aba Prefecture).A survey was made among children in the form of structured questionnaire.The questionnaire content included the social demographics (gender, age, ethnicity, household asset, educational level of parents, number of children in the family), academic performance at school (math score and absence from school in the last semester) and self-reported conditions (seizure, headache, fatigue, and nausea).The mathematical learning ability of school-aged children was evaluated using the international standard mathematical ability test (hereinafter, the mathematical test), which was performed by well-trained investigators and children completed the test by themselves within prescribed time.Serum samples were collected from children to detect the antibodies against C.cellulosae using the enzyme-linked immunosorbent assay (ELISA) method.The independent impact of C.cellulosae infection on the mathematical learning ability was analyzed with the multivariable linear regression.Results 3 041 children in our sample, and 2 682 children participated in all the questionnaire survey, the mathematical test and serological examination.The mean score of the mathematical test was 55.65 ± 17.15.The C.cellulosae seropositive rate was 6.67%(179/2 682).Among the seropositive students, 72.07%(129/179) reported headache, 37.99%(68/179) reported frequent fatigues, 5.59% (10/179) reported frequent feelings of nausea, and 2.80%(5/179) reported occurrence of seizure.129 children were seropositive accompanied by headache, whose mean score of the mathematical test was 52.33 ± 1.46, 1 648 children were seronegative accompanied by headache, whose mean score of the mathematical test was 55.94 ± 0.43, and the difference of mathematical test score in two types children was statistically significant(t = 2.28, P < 0.05).After controlling the factors of social demographics and academic performance at school, the multivariable linear regression analysis showed that the factor of seropositive accompanied by seizure, and seropositive accompanied by headache were statistically significant（β = -18.02, -4.06; P < 0.05).Besides, the household asset, the educational level of mother and the number of children in the family were relevant to the mathematical test score of children (β = 2.61, 3.86, -6.19; P < 0.01).Conclusion The epidemiological status of C.cellulosae infection among children in Tibetan agricultural areas is becoming a problem that needs more attention.C.cellulosae infection has shown detrimental effects on children’s mathematical learning ability to some extent.

Screening and evaluation of a specific nucleic acid target sequence for detection of trypanosome

Qing-hua HONG, Yun-cheng WANG, Hong-yan LI, Rui-xiang ZHANG, Jian LI, Mu-xin CHEN, Xiao LIU, Ming-bo YIN, Jing-wen WANG, Wei HU

2017, 35(6):  588-593. 

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Objective To screen and identify a specific nucleic acid target sequence for detecting Trypanosoma based on 60S ribosomal protein L44 (60S RPL44) gene sequence of Trypanosoma brucei brucei, and evaluate its sensitivity and specificity.Methods The 60S RPL44 sequence of T.b.brucei was blasted with its homologous sequences from Babesia microti, Leishmania donovani, Plasmodium vivax, P.falciparum, Toxoplasma gondii, as well as the other genera of protozoan and trypanosome species, on a public database.Meanwhile, several pairs of primers for 60S RPL44 of T.b.brucei were designed using Primer Premier 6, of which one pair was selected for screening specific nucleic acid target sequence of Trypanosoma according to the sequences alignment and primers score result.Furthermore, the specificity of the primers was evaluated among DNA from T.b.brucei, T.b.rhodesiense, T.congolense, T.evansi and the above-mentioned other five genera of protozoan parasites.The sensitivity was assessed using gradient concentrations of T.evansi genomic DNA (10 ng/μl, 1 ng/μl, 100 pg/μl, 10 pg/μl and 1 pg/μl).Results A pair of primers for detecting Trypanosoma based on T.b.brucei 60S RPL44 sequence were selected: primer P1: 5′-CCTGATGAAAGGTGCAATG-3′; primer P2: 5′-CGTTTTCGCCTTCTTGTGGA-3′.This pair of primers could amplify T.b.brucei, T.brucei rhodesiense, T.congolense and T.evansi DNA with a product of 156 bp, rather than Babesia microti, Leishmania donovani, Plasmodium vivax, P.falciparum and T.gondii.The sensitivity in detecting T.evansi genomic DNA was 10 pg/μl.Conclusion A highly sensitive and specific nucleic acid target for detection of trypanosome is found.

INFORMATION REPORT

Risk factors for malaria: a Meta-analysis

Xu-can ZENG, Chun-hai LUO, Zu-rui LIN, Zi-you ZHOU, Quan LV, Xing-wu ZHOU, Jian-xiong LI, Yao-wu ZHOU, Xiao-dong SUN

2017, 35(6):  594-597. 

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To analyze the risk factors for malaria, case-control studies on malaria risk factors from 1990 to 2016 were collected from databases of PubMed, CNKI, EMbase and Cochrane.The odds ratio (OR) and 95% confidence interval (95%CI) were calculated for each factor.Meta-analysis was performed with the RevMan 5.2 software.Eighteen case-control studies involving 3 509 cases and 12 447 controls were finally included for the analysis.Meta-analysis results revealed risk factors of never or rarely using a mosquito net, housing structure for the temporary use, exposure to the endemic areas 1 month before malaria onset, malaria occurrence in family members or neighborhood in the past year, with pooled OR values (95%CI) of 1.62 (1.42,1.85), 1.57 (1.09,2.28), 2.88 (2.00,4.16), and 3.59 (2.93,4.40), respectively (all P < 0.05).The results suggest the importance of paying attention to populations who have contacts with patients and those with an epidemiological history, and strengthenting the health education of personal protection and so on.

REVIEWS

Endemic status of schistosomiasis in floating population and challenges in schistosomiasis control in China

Zhou GUAN, Shan LV, Shi-zhu LI, Jing XU

2017, 35(6):  598-603. 

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The task of schistosomiasis prevention and control is transforming from control to elimination.Because of the social development and frequent economic trade activities, the effect of floating population on schistosomiasis control is becoming evident.This paper reviews the endemic status and characteristics of schistosomiasis control in floating population in China.The challenges of schistosomiasis control and prevention in floating population are analyzed, and some suggestions are raised for future implementation of control measures, in order to provide new information for managerial staff and technical personnel of schistosomiasis control.

Epidemic status of HIV/AIDS with intestinal protozoa infection

Xue-jiao TENG, Jia-xu CHEN, Li-guang TIAN

2017, 35(6):  607-614. 

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Intestinal parasite infection in HIV/AIDS can accelerate disease progression, and is a main cause for chronic diarrhea among the HIV-positive individuals, even leading to death in severe cases.At present, HIV/AIDS with intestinal protozoa infection is a largely neglected public health problem, with underestimation of its harmful consequences.This paper reviews the epidemic status of infection with common intestinal protozoa Blastocystis hominis, Cryptosporidium spp., Giardia lamblia, and Entamoeba histolytica among HIV/AIDS individuals in areas with different economic levels worldwide, in order to provide reference for the control of HIV/AIDS with intestinal protozoa infection.

Effects of endoplasmic reticulum stress in parasite infection

Qi-pei WEI, Yong-fen QI, Yan-rong YU

2017, 35(6):  617-622. 

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Endoplasmic reticulum stress (ERS) is a disorder of endoplasmic reticulum homeostasis caused by excessive secretion of proteins or protein misfolding under infection and other stressors.ERS triggers a series of signaling and transcriptional events, termed unfolded protein response (UPR).Although UPR can restore endoplasmic reticulum homeostasis by reducing protein translation, aiding protein folding and degrading misfolded protein, long-term, serious ERS causes apoptosis of stressed cells and local inflammation.During infection of parasites, ERS is induced in host cells, together with initiation of UPR.The parasites can use this reaction in various ways to promote its survival and pathogenicity, and to influence inflammation, oxidative stress, apoptosis and immune responses of host cells.This review summarizes research progress on the effects of ERS in parasite infection, in the aim to provide new clues and ideas for investigating the relationship between parasites and host, and the control of parasitic diseases.

SHORT COMMUNICA TIONS

Current status and influencing factors of Clonorchis sinensis infection in rural areas of Ninghai County in Zhejiang Province

Min-xia GU, Yao-jun YU, Jin-chuan ZHANG, Bin WANG, Yi-jiang YU, Cong-han REN

2017, 35(6):  585-587. 

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This study aimed to understand the current status of Clonorchis sinensis infection in Ninghai County, the place of infection, and the knowledge and practice among the general population in Ninghai County, in order to evaluate the spread of the disease.Five towns namely Yuelong, Chalu, Shenzhen, Liyang, and Huchen were randomly selected for survey.One administrative village with streams nearby was selected from each of the towns for sampling feces from humans aged above 3 and from dogs.The modified Kato thick smear method was used to examine eggs of C.sinensis.The second intermediate host fish (Pseudorasbora parva) was also sampled to examine metacercaria in muscles on the back using the tabletting method.The knowledge and practice survey was performed on 30 randomly selected persons in each village.The results showed that among 1 034 human and 25 dog feces in 5 villages, no C.sinensis eggs were found.Of the 381 P.parva collected, the metacercariae rate was 13.39% (51/381).The knowledge and practice survey was performed on 150 people, and 145 valid questionnaires were returned.The awareness rate of clonorchiasis prevention was 13.10% (19/145), 46.21% (67/145) reported separate use of raw and cooked dishes, 7.59% (11/145) usually ate raw, half-cooked fish and shrimp, 4.14% (6/145) expressed the willingness to take a try of raw fish, 82.76% (120/145) would choose to buy anthelmintics if infected, and 2.67% (4/145) would not quit the raw or half-raw fish or shrimp diet after remedy of the disease.These results imply a predisposition to infection from P.parva, a low awareness rate of C.sinensis infection among the general population, and a high risk of infection arising from lifestyles.Therefore, healthy diet and lifestyle should be advocated.

Analysis of factors for early reporting of imported malaria cases

Xin ZHU, Yun-yun ZHANG, Zhi-guo YANG, Cheng-yun YANG

2017, 35(6):  603-606. 

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From 2012 to 2016, a total of 159 imported malaria cases were reported, predominated by falciparum malaria (76.1%, 121/159).The average time interval from disease onset to diagnosis was 4.07±6.74 days.Sixty-one cases (38.4%) first visited medical units, while 66.7% (106/159) were diagnosed in CDCs, and 64.8% (103/159) visited hospitals after disease onset.Univariate analysis identified the way of going abroad, a history of malaria, the origin of imported malaria, medical unit of first visit, the way of case-finding, and malaria onset type as factors for early reporting of malaria.Multivariate analysis identified the place origin of imported malaria, medical unit of first visit, the way of case-finding as factors for early reporting of malaria.

Investigation on human Ascaris infection in Qinghai Province in 2015

Xiao MA, Cun-zhe ZHAO, Jing-xiao* ZHANG, Yu-fang LIU, Shi-lei CHENG, Pei-yun LIU, Xue-fei ZHANG, Jun-ying MA, Yong-shun WANG, Na LIU, Wen LEI, Wei WANG, Jia LIU, Xiong-ying ZHANG, Pei-zhen ZHAN

2017, 35(6):  614-616. 

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The aim of this study was to further understand the status of Ascaris infection in humans and facilitate effective control of ascariasis in Qinghai Province.The cluster-stratified sampling method was used to detect worm eggs in 53 investigation points in 17 counties(cities) from May to September, 2015.The intestinal helminth eggs in fecal samples from residents were detected by the Kato-Katz thick smear method（one sample for three tests）, and results were analyzed by SPSS 22.0 software.A total of 13 660 persons were examined, and 140 were infected with roundworms (infection rate 1.05%), comprising of 133（95.00%） light infections and 4（5.00%） moderate infections.No heavy infection was found.The prevalence of Ascaris was higher in Ping′an （2.49%） and Minhe（2.13%） than in other counties （χ² = 70.924, P < 0.05）.The population of 0-10 years showed higher infection rate than other age groups（P < 0.05）.The Han population showed higher infection rate than other ethnicities（P < 0.05）.The preschool children had higher infection rate than other occupation groups （P < 0.05）.Unfertilized Ascaris eggs accounted for 99.29% (139/140), and only one case was found with fertilized eggs in Gonghe county.The results indicate that both prevalence and intensity of Ascaris infection are low in Qinghai.

Modification of axenic culture medium and morphological observation of Blastocystis hominis in vitro

Jyh-wei SHIN, Chen-Chieh LIAO, De-jun LIAO, Xu-ning SUN, Xiao-yin FU, Deng-yu LIU

2017, 35(6):  623-625. 

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The Locke’s egg serum (LES) biphasic medium was modified by agar replacement of protein.A sterile in vitro culture system for Blastocystis hominis was then established using LES, modified LES (mLES) and liquid-state and solid-state Iscove’s Modified Dulbecco’s Medium (IMDM).Morphological and growth characteristics of the parasites in different media were observed.Blastocystis hominis showed vacuolar and granular morphologies in all types of media and had the most rapid growth in the mLES, in which the log phase was achieved on day 2 of culture (3.09 × 10⁶ parasites).The doubling times were (49.72 ± 1.35) h in LES, (24.16 ± 2.53) h in mLES, and (36.30 ± 1.50) h in IMDM.The sterile in vitro culture system for Blastocystis hominis is established using LES, mLES, and liquidus and solid-state IMDM.

Risk assessment of schistosome infection after transmission interruption based on the fuzzy comprehensive evaluation method

Xiao-jun MENG, Sheng-hua ZONG, Dong-lin GAO, Xuan ZHANG, Yan-hua QIAN, Bing LU

2017, 35(6):  626-628. 

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A risk assessment index system after schistosomiasis transmission interruption in Wuxi city was established by the Delphi method and transmission risk was assessed by the fuzzy comprehensive evaluation method. The risk assessment index system had primary indicators of natural environment, key populations and social environment, with normalized weights of 0.370 6, 0.292 9 and 0.336 5, respectively. Among the 15 secondary indicators secondary indicators, population from endemic areas of schistosomiasis had the highest combined weight of 0.125 2 while free-range livestock had the lowest combined weight of 0.037 1. The final analysis indicated a low level of schistosomiasis transmission risk after the interruption of transmission in Wuxi city, with a risk assessment score of 0.193 6.