Table of Content

30 April 2020, Volume 38 Issue 2

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SPECLAL REPORT

Epidemiological characteristics of malaria and progress on its elimination in China in 2019

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

2020, 38(2):  133-138.  doi:10.12140/j.issn.1000-7423.2020.02.001

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Malaria epidemic data from 31 provinces/municipalities/autonomous regions (Taiwan region, Hong Kong and Macao not included) of China in 2019 were collected through the Malaria-specific Reporting System in the Information Network for Parasitic Diseases Control. The epidemiological characteristics of malaria and progress on its elimination were analyzed. In 2019, a total of 2 674 malaria cases were reported from 779 institutions in China, being 4 cases less than that reported in 2018(2 678 cases); 2 673 cases were reported as imported cases, and 1 case was malariae malaria with a long incubation. No indigenous cases were reported. The total reported cases included 2 487 Chinese cases (93.0%, 2 487/2 674) and 187 cases of foreign nationality (3.0%, 187/2 674); with a male-to-female ratio of 14.9 : 1, and a age distribution of mostly at 30-49 years (60.3%, 1 613/2 674). The reported cases comprised vivax malaria (10.8%, 289/2 674), falciparum malaria (72.9%, 1 950/2 674), malariae malaria (3.6%, 97/2 674), ovale malaria (11.1%, 298/2 674), mixed infections (1.3%, 35/2 674) and clinically diagnosed cases (0.2%, 5/2 674). The cases were reported from 29 provinces, with the top 5 provinces being Jiangsu (9.1%, 244/2 674), Shandong (8.5%, 228/2 674), Henan (8.5%, 227/2 674), Guangdong (7.7%, 206/2 674) and Sichuan (7.4%, 199/2 674), among them, 433 cases (16.2%, 433/2 674) were reported from 4 border provinces (Yunnan, Guangxi, Liaoning, Xinjiang). Totally 19 deaths (0.3%, 19/2 674) were reported from 11 provinces, increased by 171.4% compared to the 7 deaths in 2018. The 1-3-7 approach was implemented nationwide with good performance: all cases were reported within 24 hours after diagnosis, 97.9% (2 619/2 674) of them were investigated on epidemiology within 3 days, and 2 326 foci were identified, investigated and responded to within 7 days. In conclusion, no indigenous malaria cases have been reported since 2017, more efforts should be made to continuously strengthen surveillance and management for imported malaria and border malaria, thus to prevent from re-transmission, reduce the risk of death and consolidate the achievements of malaria elimination in the country.

ORIGNAL ARTICLES

Regulations of immune responses by artesunate in combination with rIL-33 in the treatment of cerebral malaria in mice

Yun-ting DU, Wei ZHAO, Ya-ming CAO, Lan XU

2020, 38(2):  139-145.  doi:10.12140/j.issn.1000-7423.2020.02.002

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Objective To investigate the effects of artesunate (ART) in combination with recombinant interleukin-33 (rIL-33) in the treatment of cerebral malaria in mice infected with Plasmodium berghei ANKA (PbA) and its effects on immune responses of mice.Methods Forty female C57BL/6 mice were randomly divided into 5 groups (n = 8): PbA infection without treatment (PbA group), PbA infection with treatment (PbA+rIL-33 group, PbA+ART group, and PbA+rIL-33+ART group), and normal control group. Mice in the PbA infection groups were injected intraperitoneally (i.p.) with 1 × 10 ⁶ PbA-infected red cells. Two to four days post-infection, mice in the PbA+rIL-33 group received daily i.p. injections of rIL-33 (0.2 μg/mouse) for 3 consecutive days, those in the PbA+ART group received 40 mg/kg ART (once daily for 3 days) by gavage, and those in the PbA+rIL-33+ART group received ART and rIL-33 injection for 3 consecutive days. The PbA group received no treatment. The control group received the same volume of PBS at the same time points as above. From day 3 after infection, tail vein blood was collected every other day, blood smears were made for Giemsa staining to examine the infection rate of red cells, and the death and survival time span were recorded as well. To assess the integrity of the blood-brain barrier (BBB), on day 5 after infection, evans blue solution was given to the mice intravenously, then the brain tissue eluent was examined to detect penetrated blue dye passing through the barrier by measuring absorbance at 630 nm (A₆₃₀). On day 5 after infection, four mice of each group were sacrificed to determine the percentage and absolute number of Th1/Th2, Tregs, macrophages and Toll-like receptor 4 (TLR4) cells in spleen by flow cytometry. Results In the PbA group, neurological symptoms first appeared on day 6, and deaths occurred from day 6 to day 13, while in the PbA+rIL-33 and the PbA+ART groups deaths occurred on day 8. The neurological symptoms of the PbA+rIL-33+ART group were significantly suppressed, and deaths occurred on day 12 post infection. Two of 7 mice died of anemia on day 23 post infection. The infection rate of red cells in the PbA+rIL-33+ART group increased from 0.30% to 19.67% during day 3 to 13 after infection, remaining consistently lower than those of the PbA group (0.93%-20.00%) and PbA+rIL-33 group (0.46%-19.67%) (P < 0.05). In addition, BBB integrity assessment showed that the A₆₃₀ in the PbA+rIL-33+ART group was (0.11 ± 0.01), significantly lower than those in the PbA group (0.44 ± 0.01) (P < 0.01), the PbA+rIL-33 group (0.19 ± 0.01) (P < 0.01) and the PbA+ART group (0.27 ± 0.02) (P < 0.01). Flow cytometry assay showed that the percentage of Th1 cells in the PbA+rIL-33+ART group was (7.51 ± 0.26)%, which was significantly lower than that in the PbA group [(14.27 ± 0.91)%, P < 0.01], but did not differ significantly from those in the PbA+ART group [(9.56 ± 1.75)%] and PbA+rIL-33 group [(8.67 ± 0.26)%] (P > 0.05). The percentage of Th2 cells in the PbA+rIL-33+ART group was (2.63 ± 0.27)%, which was significantly higher than those in the PbA group [(0.80 ± 0.13)%, P < 0.01] and the PbA+ART group [(0.88 ± 0.12)%, P < 0.01], but did not differ significantly from that in the PbA+rIL-33 group [(1.70 ± 0.54)%] (P > 0.05). The percentage of macrophages in the PbA+rIL-33+ART group was (3.85 ± 0.32)%, which was significantly higher than that in the PbA group [(2.89 ± 0.89)%] (P < 0.05), but did not differ significantly from those in the PbA+ART group [(3.15 ± 0.46)%] and the PbA+rIL-33 group [(4.11 ± 0.68)%] (P > 0.05). The percentage of Treg cells in the PbA+rIL-33+ART group was (11.05 ± 1.50)%, which was significantly higher than those in the PbA group [(6.44 ± 0.09)%, P < 0.05] and the PbA+ART group [(6.27 ± 0.39)%, P < 0.01], but did not differ significantly from that in the PbA+rIL-33 group [(9.34 ± 0.61)%] (P > 0.05). The percentage of TLR4 cells in the PbA+rIL-33+ART group [(1.43 ± 0.21)%] was significantly higher than that in the PbA group [(3.76 ± 0.41)%] (P < 0.01), but did not differ significantly from those in the PbA+ART group [(1.69 ± 0.26)%] and the PbA+rIL-33 group [(1.61 ± 0.15)%](P > 0.05).Conclusion rIL-33 combined with ART can protect the mouse brain from experimental cerebral malaria damage, and improve the therapeutic outcome through balancing Th1/Th2 immune responses.

The mutation polymorphism of G6PD gene coding region in vivax malaria patients in Yunnan Province, China

Shu-ping LIU, Ying DONG, Yan-chun XU, Yan LIU, Yan DENG, Cang-lin ZHANG, Meng-ni CHEN

2020, 38(2):  146-151.  doi:10.12140/j.issn.1000-7423.2020.02.003

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Objective To analyze the polymorphism of G6PD (glucose-6-phosphate dehydrogenase) gene mutation in coding regions and its correlations with the proneness to primaquine hemolysis in vivax malaria patients in Yunnan Province, China.Methods Blood samples were collected from the vivax malaria patients receiving an treatment regime of “eight-day therapy of chloroquine/primaquine” in Yunnan Province in 2018. Patients with “tea-colored” urine and reduced G6PD enzyme activity during the primaquine therapy were defined as primaquine-induced acute hemolysis. Human genomic DNA was extracted from the blood samples, and of the G6PD gene fragments containing 12 exon were amplified by PCR and sequenced. The sequences were compared with the nonmutation-type sequence, edited and spliced by DNAStar 11.0 and BioEdit 7.2.5 softwares to generate the cDNA sequence of G6PD gene. MEGA 5.04 and DnaSP 5.10 softwares were used to analyze the mutation polymorphism the selection effect of the cDNAs. The correlation coefficient (r) and odds ratio (OR) for primaquine-induced hemolysis was estimated.Results A total of 184 blood samples were collected, of them 44 generated complete G6PD cDNA strand sequence (length, 1 545 bp), including one sample from a hemolysis case. Alignment of the cDNA chain with the nonmutation-type sequence revealed the mutation sites including c.461 T > A, c.574 C > T, c.786 C > T, c.1059 C > T, c.1311 T > C, and c.1376 G > T, with a frequency of 1.5/100 000, 1.5/100 000, 1.5/100 000, 1.5/100 000, 45.6/100 000 and 1.5/100 000, respectively. The two-site mutation linkage between c.1311 and c.1376 was only present in the genome of the hemolysis cases, and the c.1376 site mutation was positively correlated with the primaquine-induced hemolysis (r = 1.000, P > 0.05). The 44 cDNA strands were defined into 6 haplotypes (Hap_1-Hap_6), with an expected heterozygosity, nucleic acid diversity index π, and Ka/Ks ratio were 0.493, 0.001, and 0.062, respectively. The Hap_2 type containing c.1311 single-site mutation accounted for the highest proportion of (68.2%, 30/44). The site c.1311 with high frequency of mutation had the most complex allele composition, comprising 5 types of zygote including nonmutation hemizygote (9/44, 20.4%), mutant hemizygote (16/44, 36.4%), nonmutation homozygote (5/44, 11.5%), mutant heterozygote (10/44, 22.7%), and mutant homozygote (4/44, 9.1%). The OR value of c.1311 mutation to the primaquine-induced hemolysis was 0.6879 (P > 0.05), showing no correlation in between. The D value of Tjima’s neutral test was -1.414 (P > 0.05), and the D value of the whole fragment was < 1, indicating that the mutations detected in the sequence were not a non-neutral mutation under directional selection pressure; meanwhile the Ka/Ks ratio was 0.062, indicating that the sequence was highly conservative and the frequency of missense mutation was lower than that of synonymous mutation.Conclusion In malaria patients in Yunnan Province, the T > C base substitution at c.1311 is a common mutation in G6PD coding region, but only the G > T mutation at c.1376 is positively correlated with the occurrence of oxidant induced hemolysis.

Performance comparison of three methods in detecting asymptomatic malaria infection on China-Myanmar border

Xiao-xiao WANG, Hui-hui XIAO, Fang HUANG, Shui-sen ZHOU

2020, 38(2):  152-158.  doi:10.12140/j.issn.1000-7423.2020.02.004

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Objective To compare the performances of three methods in detecting asymptomatic malaria infection in residents, and assess the prevalence of asymptomatic malaria on China-Myanmar border.Methods Cross-sectional survey was carried out in Nabang town and Zhina town in Yingjiang county of Yunnan Province, China and a resettlement site in Laiza of Myanmar. Finger-prick blood samples were collected to prepare thick/thin blood smears and dried blood spots on filter paper. Malaria parasite infection was examined by light microscopy, real-time fluorescent PCR and ultrasensitive PCR (usPCR).Results A total of 387 blood samples were collected. Light microscopy found 6 participants with asymptomatic malaria infection (5 Plasmodium vivax infection and 1 P. falciparum infection), with a detection rate of 1.6%; real-time fluorescent PCR revealed 13 asymptomatic malaria infection cases (12 P. vivax infection; 1 P. falciparum infection) with a detection rate of 3.4%; and usPCR found 38 asymptomatic malaria infection cases(29 P. vivax infection and 9 P. falciparum infection) with a detection rate of 9.8%. Using light microscopy as the gold standard, the sensitivity and specificity of real-time fluorescent PCR were 98.2% and 100%, and those of usPCR were 91.6% and 100%, respectively. The usPCR results revealed that the detection rate of asymptomatic malaria infections was highest in Nabang town (17.1%, 22/129), followed by the Laiza resettlement site (10.0%, 11/110) and Zhina town (3.4%, 5/148), showing significant differences between the three sites (P < 0.05). Among the parasite species detected, P. vivax accounts for 76.3%, while P. falciparum for 23.7%. The detection rate was higher in females (10.7%, 23/215) than in males (8.7%, 15/172), but with no significant difference (P > 0.05). The age distribution showed that, the detection rate of asymptomatic infection was highest in the examinees aged 15-29 years(17.5%, 10/57), but there were no significant differences between the age groups.Conclusion Of the three methods compared for the ability in detection of asymptomatic malaria infection, the usPCR method presents higher detection rate than light microscopy and real-time fluorescent PCR. There are a certain proportion of asymptomatic malaria infection cases among the residents on China-Myanmar border.

Bioinformatics analysis and enzymatic activity test of quiescin sulfhydryl oxidase from Plasmodium berghei

Wen-qi ZHENG, Xue-min FENG, Ya-ming CAO, Yan-qiu HAN, Jun-rui WANG

2020, 38(2):  159-165.  doi:10.12140/j.issn.1000-7423.2020.02.005

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Objective To clone and express the gene of Plasmodium berghei quiescin sulfhydryl oxidase (PbQSOX), purify the recombinant protein rPbQSOX, and analyze its bioinformatics characteristics and enzymatic activity.Methods The signal peptides, protein domains, active sites and homology of PbQSOX protein were analyzed by BLAST, SMART and ClustalW softwares. The phylogenetic tree of PbQSOX was constructed by BLAST and MAGA5. The tertiary structure of PbQSOX protein was predicted by SWISS-MODEL. Female BALB/c mice were intraperitoneally injected with 1 × 10 ⁷ P. berghei protozoan/mouse. On day 4 after infection, P. berghei genomic DNA was extracted. The PbQSOX gene was amplified by PCR. After confirming the product by sequencing, the target product was cloned into pET30a(+) vector to construct recombinant plasmid pET30a(+)-PbQSOX. The verified plasmid was transfected into E. coli BL-21 after induction by isopropyl β-D-thiogalactoside, the bacteria culture medium was analysed underwent by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The soluble recombinant PbQSOX (rPbQSOX) was purified by Ni-triacetate agarose column, and the enzyme activity was analyzed using TCEP [Tris(2-carboxyethyl)phosphine] or DL-Dithiothreitol (DTT) as the enzyme reaction substrate. Results BLAST analysis identified homologues of PbQSOX from other species. Multiple-sequence blasting and domain analysis showed that PbQSOX contained a signal peptide, and Trx1, ψErv and Erv/ALR domains, but lacked the Trx2 domain. The PbQSOX protein contained three key CXXC activation motifs (C, cysteine; X, any amino acid): CPAC, CRNC and CNYC. The phylogenetic tree constructed by MAGA5 using the neighbor joining method showed that the PbQSOX was genetically most close to the P. yoelii QSOX (PyQSOX). SWISS-MODEL analysis showed that the tertiary structure of PbQSOX can be predicted using known Trypanosoma brucei QSOX (TbQSOX). PCR amplification of PbQSOX resulted in a specific band of 1 486 bp, sequence of which was confirmed by sequencing. The PET30a (+) -PbQSOX plasmid was verified by sequencing after Nde Ⅰ and Hind Ⅲ digestion. SDA-PAGE and Western blotting analysis showed that rPbQSOX was expressed in soluble form with a relative molecular weight of about 59 000. The enzymatic activities of purified rPbQSOX toward TCEP and DTT were 0.84 ± 0.18 and 0.78 ± 0.14, respectively.Conclusion The expressed PbQSOX protein contains a single Trx1 domain. The rPbQSOX is a soluble protein that has some catalytic activity of the quiescin sulfhydryl oxidase.

Establishment of the detection method for Schistosoma japonicum by recombinase polymerase amplification combined with electrochemical DNA biosensor

Wang-ping DENG, Bin XU, Qing-hua HONG, Sheng-lin WANG, Chao LV, Yin-long LI, Shi-ping SONG, Jun-hu CHEN, Jing XU, Shi-zhu LI, Wei HU, Xiao-nong ZHOU

2020, 38(2):  168-174.  doi:10.12140/j.issn.1000-7423.2020.02.006

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Objective To establish a sensitive, specific and simple nucleic acid detection method for Schistosoma japonicum by combining recombinase polymerase reaction (RPA) with electrochemical DNA sensor.Methods Genomic DNA of S. japonicum adult worms was extracted. Applying the SjR2 gene fragment as the target sequence, primers and probe were designed for recombinase polymerase amplification (RPA), which was then combined with electrochemical DNA sensor(EC) technique; the probe was immobilized on the multi-channel electrode chips for interfacial RPA amplification and electrochemical detection, thus establishing the isothermal detection method (RPA-EC) for S. japonicum nucleic acid. The RPA reaction conditions were optimized and the sensitivity of the RPA-EC method was tested by detecting 10 ^-4 ng, 10 ^-5 ng, 10 ^-6 ng, 10 ^-7ng and 10 ^-8 ng of S. japonicum genomic DNA. The specificity of the RPA-EC method was evaluated by detecting 10 ng of genomic DNA of S. japonicum, S. haematobium, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis. To verify the feasibility of the RPA-EC method to detect the dynamic changes of the SjR2 gene fragment in sera, 10 adult C57 mice aged 6 to 8 weeks were each infected with 40 S. japonicum cercariae by abdominal challenge, and serum samples were collected on days before infection (0 d), and 7, 21 and 35 after infection. Results The RPA-EC assay could complete the amplification and detection of SjR2 fragment within 30 min at 37 ℃, and the lower detactable limit for SjR2 fragment in adult S. japonicum genomic DNA could be 10 ^-8 ng. In addition, there was no cross-reaction with the genomic DNA of S. mansoni, S. haematobium, P. westermani, F. gigantica and C. sinensis, suggesting a high specificity. It is of note, the results of animal experiments showed that the RPA-EC assay could sensitively detect the SjR2 gene fragment in the sera of mice on day 7, 21 and 35 post infection. Conclusion The RPA-EC combinational detection method established in this study exhibits high sensitivity and good specificity, and is easy to operate, showing the potential for future application.

Prevalence of visceral leishmaniasis in China in 2018

ZHOU Zheng-bin, LI Yuan-yuan, ZHANG Yi, LI Shi-zhu

2020, 38(2):  175-181.  doi:10.12140/j.issn.1000-7423.2020.02.007

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Objective To understand the prevalence of visceral leishmaniasis in China in 2018.Methods Information of visceral leishmaniasis cases in 2018 was collected from the web-based National Diseases Reporting Information System (NDRIS) operated by the Chinese Center for Disease Control and Prevention. A database was established, excluding suspected cases, duplicates and cutaneous leishmaniasis cases. The three-dimensional distribution of visceral leishmaniasis cases was descriptively analyzed using Microsoft Excel 2016.Results In 2018 a total of 180 cases of visceral leishmaniasis were reported in 78 counties of 11 provinces (autonomous regions and municipalities), mainly distributed in Gansu (66 cases), Shanxi (38) and Shaanxi Provinces (27). Forty counties were endemic regions of visceral leishmaniasis, with a total of 134 reported indigenous cases, and the other 38 counties were non-endemic regions, with a report of 46 imported cases of visceral leishmaniasis. Particularly, Zhouqu county, Dangchang county and Wudu district were the major prevalent counties with 22, 18 and 10 cases reported, respectively, accounting for 27.8% (50/180) of the total cases reported. The Huazhou district and Linwei district of Weinan City in Shaanxi Province reported re-emergence of indigenous visceral leishmaniasis cases, being recognized as the visceral leishmaniasis-reemerging regions. The occurrence of visceral leishmaniasis peaked from April to June, with a male/female ratio of 1 ∶ 0.7. Farmers and children were at a high risk of visceral leishmaniasis, accounting for 42.2% (76/180) and 34.5% (55/180) of the total, respectively. The visceral leishmaniasis cases were mainly distributed in the people at ages of ≥15 years. The age distribution varied significantly among different types of the disease, with the desert-type zoonotic visceral leishmaniasis distributed mainly in infants of age 0-2 years, and the anthroponotic and mountain-type zoonotic visceral leishmaniasis distributed mainly in farmers aged ≥15 years.Conclusion The visceral leishmaniasis is endemic at low prevalence in China, but the endemic area is extending gradually.

Evaluation of the value of K26 sequence applied in identification of Leishmania isolates in China

Jian-xiu LIU, Chun-hua GAO, Yue-tao YANG, Bin ZHENG, Jun-yun WANG

2020, 38(2):  181-187.  doi:10.12140/j.issn.1000-7423.2020.02.008

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Objective To evaluate the application of Leishmania K26 sequence in identification of Leishmania isolates in China.Methods Sixteen Leishmania isolates collected from different domestic endemic areas and 6 international reference strains were cultured for collection of promastigotes, from which DNA was extracted for amplification of K26 sequence by PCR. The K26 sequence was confirmed by sequencing. The sequence alignment was made using the ClustalX2 software. A phylogenetic tree was constructed based on the K26 sequences of highly-homologous Leishmania isolates downloaded from the GenBank database, to analyze the genetic relationship among the Leishmania isolates from different endemic areas in China.Results PCR amplification showed a specific band from all the 16 isolates and the DD8 international reference strain, while no specific band was found from five non-L. donovani complex strains. Sequence analysis indicates that all 3 isolates 801, KS-2 and KS-6 from the endemic areas of anthroponotic leishmaniasis generated a 920 bp fragment in PCR; 4 isolates XJ771, JIASHI-1, JIASHI-2 and JIASHI-5 from endemic areas of natural-sourced leishmaniasis all generated a 491 bp fragment in amplification reaction; 4 strains Cy, SC6, Hebei-Lv and Shanxi-Yang from endemic areas of zoonosis (specifically canine) leishmaniasis generated a 404 bp in amplification; 5 strains KXG-Xu, KXG-Liu, KXG-65, KXG-918 and KXG-927 from endemic areas of cutaneous leishmaniasis all generated a 449 bp fragment in amplification. Among the endemic areas, the K26 sequences of intra-area isolates showed 100% homology, while the sequences of enter-area isolates displayed a lower homology (40.2%-91.4%). Phylogenetic tree analysis showed that the 16 isolates of Leishmania in China clustered into 2 groups and 3 subgroups, among which nine isolates including KXG-Xu, KXG-Liu, KXG-65, KXG-918, KXG-927, XJ771, JIASHI-1, JIASHI-2, and JIASHI-5 clustered into subgroup A, while five isolates Cy, SC6, Hebei-Lv, Shanxi-Yang and L. infantum clustered into subgroup B, and both subgroups clustered into groupⅠ. The 801, KS-2 and KS-6 isolates clustered with L. donovani as subgroup C, which further clustered with the DD8 international reference isolate into group Ⅱ.Conclusion The K26 sequences of Leishmania can be used for identification of Leishmania isolates in China.

Analysis of miR-71 secretion and expression characteristics of Echinococcus granulosus protoscolex

Lu-jun YAN, Ya-ting LI, Jun-jun DING, Jing YANG, Ya-dong ZHENG, Yi-xia CHEN

2020, 38(2):  188-193.  doi:10.12140/j.issn.1000-7423.2020.02.009

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Objective To analyze the secretion and expression characteristics of miR-71 of Echinococcus granulosus protoscolex under stimulation by insulin, albendazole and artificial gastric fluid.Methods E. granulosus cysts in sheep liver and lung tissues were collected at a slaughterhouse in Xinjiang, China. The E. granulosus cysts were fixed, embedded and sectioned, followed by hybridization with a miR-71 probe. The sections were then incubated with antibody labeled with fluorescein isothiocyanate (FITC), and then stained with a nucleic acid dye 4’, 6-diamidino-2-phenylindole (DAPI) to visualize the allocation of miR-71 in E. granulosus protoscoleces and cysts wall with fluorescence microscopy, analyzing the distribution of miR-71 in the protoscoleces at various developmental stages, for ascertaining the expression of miR-71 in the protoscoleces. In addition, the protoscoleces were cultured in the presence of insulin, albendazole and artificial gastric fluid and the supernatant was collected to isolate exosomes by differential centrifugation. The morphology of exosomes was observed under a transmission electron microscope. The particle-size distribution of the exosomes was measured by a nanoparticle size analyzer. The enolase and 14-3-3 protein were used as biomarkers for identification of exosome by Western blotting. The abundance of miR-71 in exosomes was determined by real-time quantitative PCR (qPCR).Results In situ hybridization showed that miR-71 was expressed in the middle and late developmental stages of protoscoleces and in the germinal layer of cyst wall of E. granulosus. The exosomes had a size of 48.9 nm, 49.7 nm and 65.4 nm in the groups of artificial gastric fluid, insulin, and albendazole, respectively, all within the range of 40-120 nm, and morphologically shaped as a membrane-enveloped sphere. Western blotting detected the presence of enolase and 14-3-3 protein in exosomes, demonstrating successful extraction of exosomes. qPCR showed that the expression level of miR-71 in exosomes secreted from protoscoleces in the test group with artificial gastric fluid, insulin, and albendazole was 1.84, 1.87 and 2.38 folds of the control group (t = 12.8, 26.7 and 29.3, P < 0.01), respectively.Conclusion miR-71 is widely expressed in protoscoleces and cyst wall of E. granulosus, and could be secreted via exosomes. The expression and secretion of miR-71 can be upregulated by stimulations with insulin, artificial gastric fluid and albendazole.

Expression, localization and preliminary evaluation of diagnostic value of Echinococcus granulosus phosphoglycerate mutase

Hong-yu SONG, Yu-qing LIANG, Rui-qi HUA, Yuan SHI, Ai-guo YANG, Li GUO, Dong-bo YUAN, Yue XIE, Guang-you YANG

2020, 38(2):  194-201.  doi:10.12140/j.issn.1000-7423.2020.02.010

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Objective To clone and express Echinococcus granulosus phosphoglycerate mutase (EgPGAM), analyze its immunoreactivity and distribution in protoscoleces, hydatid cysts and adult worms, and preliminarily evaluate the diagnostic value.Methods Protoscolex RNA was extracted and reverse transcribed to cDNA, from which EgPGAM gene was amplified by PCR and sequenced. The physicochemical properties, conserved domains, antigen epitopes and homology of EgPGAM were analyzed by bioinformatics softwares. Mega 5.0 software was used to analyze the characteristics of the sequence, and the phylogenetic tree was constructed with the neighbor joining method. The EgPGAM gene was inserted into the pET32a plasmid, and transferred into Escherichia coli BL21(DE3) for induction of expression. The expressed proteins were purified by nickle column, and further analyzed by SDS-PAGE for verifying the expression products, and Western blotting for immunoreactivity. The distribution of EgPGAM in protoscoleces, hydatid cysts and adults was analyzed by immunofluorescence assay. The diagnostic value of EgPGAM was evaluated by indirect enzyme-linked immunosorbent assay(ELISA).Results The EgPGAM gene is 756 bp in length, encoding 251 amino acids. The encoded protein has a relative molecular weight Mr about 28 000, an isoelectric point of 8.29, containing no signal peptide or transmembrane region. EgPGAM protein contained 12 highly-conserved amino acid sites, 3 conserved catalytic sites, one substrate-binding site and 7 B epitopes. The EgPGAM had 99%, 94%, 78%, 57%, 45% and 4% amino-acid sequence homology with PGAMs of E. multilocularis, Taenia solium, Clonorchis sinensis, Caenorhabditis elegans, Homo sapiens and Ovis aries, respectively. SDS-PAGE showed that EgPGAM was highly expressed in E. coli BL21(DE3), mainly in soluble form. Western blotting showed reactions of EgPGAM with serum samples from E. granulosus-infected sheep. Immunofluorescence assay revealed that the EgPGAM was localized mainly on the tegument and hooks of protoscoleces, the germinal layer of the hydatid cyst and the cyst wall parenchyma of adults. The sensitivity and specificity of EgPGAM-based indirect ELISA were 55.6%(15/27) and 86.4% (89/103), respectively.Conclusion EgPGAM was widely distributed in the larvae and adult worms of E. granulosus. The recombinant EgPGAM had apparent immunoreactivity, but with low sensitivity in the indirect ELISA test. Therefore, EgPGAM is not a suitable candidate of diagnostic antigen for echinococcosis.

LncRNA 017418 and cytokine expression in mice immunized with Echinococcus granulosus rP29

Chan WANG, Song-hao YANG, Fei DONG, Xian-cai DU, Ji-hui YANG, Nan NIU, Ming-xing ZHU, Hao WANG, Ya-na WANG, Wei ZHAO

2020, 38(2):  201-206.  doi:10.12140/j.issn.1000-7423.2020.02.011

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Objective To preliminarily investigate the expression of long-chain non-coding RNA 017418 (lncRNA 017418) and cytokines during immune response of mice induced by recombinant protein P29 (rP29).Methods The recombinant fusion expression strain of Escherichia coli pET28a-P29/BL21 was incubated with LB liquid medium, to which isopropyl-β-D-thioglycoside (IPTG) was added to induce protein expression. The purified recombinant protein rP29 was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thirty-six female BALB/c mice were randomly divided into the control group, adjuvant group and immunization group (12 mice each). To immunization group mice, 10 μg purified rP29 was mixed with an equal volume of Freund’s complete adjuvant forming emulsion, and injected subcutaneously at three points at abdomen at a total volume of 100 μl/mouse. The adjuvant group was injected with an equal volume of Freund’s complete adjuvant alone, while the control group received no treatment. Two weeks after the first immunization, the same volume of booster with incomplete Freund’s adjuvant was given once. Two weeks after boosting, spleen cell suspensions were prepared in sterile condition and lymphocytes were separated. Flow cytometry was performed to assay the lymphocyte subsets CD4 ⁺ T, CD8 ⁺ T and B lymphocytes. The expression of lncRNA 017418, Notch1, interleukin-2 (IL-2), interferon gamma (IFN-γ) and IL-10 in lymphocytes was detected by real-time fluorescence quantitative PCR (qRT-PCR). Data were analyzed with SPSS 17.0 and GraphPad Prism 6.0 softwares. Results Produced by IPTG induction and affinity chromatography purification, the purified rP29 showed a relative molecular weight of ~31 000. qRT-PCR revealed that the relative expression of lncRNA 017418 in spleen lymphocytes in the immunization group was 2.25 ± 0.10, which was significantly higher than those in the control (0.81 ± 0.05) and adjuvant groups (0.99 ± 0.13) (P < 0.01). The proportions of CD4 ⁺ T, CD8 ⁺ T and B lymphocytes found by flow cytometry were 22.0%, 9.2% and 59.8% in the control group; 22.8%, 7.6% and 60.7%in the adjuvant group; and 22.1%, 9.7% and 60.4% in the immunization group, respectively. The relative expression of lncRNA 017418 in CD4 ⁺ T lymphocyte in the immunization group was 1.49 ± 0.03, significantly higher than those in the control group (0.97 ± 0.02) and the adjuvant group (1.06 ± 0.10) (P < 0.01); those in the CD8 ⁺ T cells in the three groups were 1.87 ± 0.12, 2.06 ± 0.14, and 2.00 ± 0.09, respectively (P > 0.05); and those in the B cells were 1.03 ± 0.03, 0.97 ± 0.07, and 1.08 ± 0.12, respectively (P > 0.05). The relative expression of Notch1 in the CD4 ⁺ T lymphocytes was 1.92 ± 0.04 in the immunization group, higher than those in the control group (1.10 ± 0.10) and adjuvant group (1.20 ± 0.08) (P < 0.01). The relative expression of IL-2 was 1.45 ± 0.07 in the immunization group, higher than those in the control group (1.07 ± 0.08) and adjuvant group (1.09 ± 0.118) (P < 0.01). The relative expression of IFN-γ was 1.51 ± 0.11 in the immunization group, higher than those in the control group (0.92 ± 0.05) and adjuvant group (1.03 ± 0.14) (P < 0.01, P < 0.05). The relative expression of IL-10 was 0.52 ± 0.01 in the immunization group, lower than those in the control group (0.95 ± 0.04) and adjuvant group (1.05 ± 0.10) (P < 0.01). There is no statistically significant difference found between the control group and the adjuvant group(P > 0.05). Pearson correlation coefficient analysis showed that lncRNA 017418 was positively correlated with Notch1, IL-2, and IFN-γ (r = 0.824 4, 0.590 2, 0.841 5; P < 0.01), and negatively correlated with IL-10 (r = -0.443 2; P < 0.05). Conclusion LncRNA 017418 expression is up-regulated in rP29-immunized mice; the expression of Notch1, IL-2 and IFN-γ are up-regulated in CD4 ⁺ T lymphocytes; and IL-10 expression is down-regulated in CD4 ⁺ T lymphocyte.

Application and verification of auxin-inducible degron system in Toxoplasma gondii Chinese type 1 strain

Ai-xia YAN, Yong-gen JIA, Yang ZOU, Jing-jing LI, Min-jun HUANG, Jun-chao GU

2020, 38(2):  207-212.  doi:10.12140/j.issn.1000-7423.2020.02.012

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Objective To regulate the expression of fluorescent proteins in Toxoplasma gondii Chinese type 1 strain using the auxin-inducible degron (AID) system.Methods The nucleotide sequence of plant auxin receptor-transport inhibitor response protein 1 (TIR1) fused with a FLAG tag at the 3′ end (TIR1-FLAG) was synthesized, and ligated into the pTub-eGFP-CAT vector to obtain the pTub-TIR1-FLAG-CAT expression plasmid. The plasmid was transfected into the Toxoplasma gondii Chinese type 1 strain by electroporation. After chloramphenicol screening and limiting dilution, positive clones were selected, in which the inserted TIR1 gene and its expression was examined by PCR and Western blotting. The verified strain clone with correct insertion was designated as the TIR1 strain. The plasmid pTub-mCherry-Ty-AID-DHFR expressing the mCherry-Ty-AID reporter gene was constructed and integrated into the genome of the TIR1 strain by electroporation, from which a clone capable of stable expression of mCherry-AID protein was obtained by pyrimethamine screening and ultimate limiting dilution, and designated as TIRI:mCherry-AID strain. The TIR1:mCherry-AID parasites were divided into the regulating group and control group, and cultured with an auxin, indole-3-acetic acid (IAA) solution (500 μmol/L) or an equal volume of ethanol for 3 h accordingly. Then the expression of mCherry fluorescent protein was detected by indirect immunofluorescence assay (IFA) and Western blotting using anti-TgGAP45 and anti-Ty-1 antibodies.Results The PCR amplification showed a specific amplification band at 1 816 bp. Western blotting showed that the anti-FLAG-Tag antibody can recognize a protein band with a relative molecular mass (M_r) of 70 000 in the TIR1 strain, and anti-Ty-1 antibody can detect a specific band with M_r of about 58 000 in the TIR1:mCherry-AID strain, which equales to the theoretical size of mCherry-Ty protein fused with AID at the C terminus; however, no specific band was detected in the auxin/IAA group. IFA assay indicated that red fluorescent protein was seen in the TIR1:mCherry-AID in the control group, but no mCherry protein was detected in the auxin/IAA group.Conclusion The AID system can regulate mCherry fluorescent protein expression in T. gondii Chinses 1 strain.

Galectin-1 inhibits Th2 inflammatory responses in mice with allergic asthma

Min ZHI, Fen-xuan ZHOU, Xin GUAN, Jian-wen ZHONG, Xiang-qian LUO

2020, 38(2):  213-218.  doi:10.12140/j.issn.1000-7423.2020.02.013

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Objective To investigate the mechanisms underlying the inhibitory effects of galectin-1 on Th2 inflammatory responses in a mouse model of allergic asthma.Methods Fifty-four female BALB/c mice were randomized into control group, ovalbumin (OVA) group, and OVA+galectin-1 treatment group. The mice in the OVA and treatment groups received subcutaneous injections of sensitizer solution [containing 100 μg of OVA in 10% Al(OH)₃] on days 0, 3 and 7, while those in the control group were injected with same volumes of saline. Seven days later, the OVA and treatment groups were challenged by 50 μg of intranasal OVA, while the control group was given 50 μl of saline, once daily for 7 consecutive days. Two hours after each challenge, intranasal galectin-1(1 μg/ml) was applied to the treatment group, while the control and OVA groups were given 10 μl of saline. After completion of treatment on day 22, the airway hyperresponsiveness was examined. Bronchoalveolar lavage fluid (BALF) was collected for Giemsa’s staining to classify and count different inflammatory cells. Lung tissue sections underwent hematoxylin-eosin staining to assess inflammatory pathology. ELISA was performed to analyze protein levels of allergen-specific IgE, Th2 inflammatory cytokines (IL-4, IL-5, and IL-13), IFN-γ, IL-10 and TGF-β in orbital blood serum. Splenic lymphocytes were collected to detect the proportion of regulatory T (Treg) cells by flow cytometry.Results The airway hyperresponsiveness of the OVA+galectin-1 treatment group (Penh value, 2.08 ± 0.17) was significantly lower than that of the OVA group (5.77 ± 0.64) (P < 0.05). The numbers of eosinophils, macrophages and neutrophils in the treatment group were (1.00 ± 0.54) × 10 ⁴/ml, (6.00 ± 0.98) × 10 ⁴/ml and (2.00 ± 0.41) × 10 ⁴/ml, respectively, which were significantly different from those in the OVA group [(3.00 ± 0.04) × 10 ⁴/ml, (4.60 ± 0.55) × 10 ⁴/ml and (2.20 ± 0.30) × 10 ⁴/ml] (P < 0.05). HE staining showed inflammatory cell infiltration and airway wall thickening around the bronchus in the OVA group, while the pulmonary inflammation was significantly ameliorated in the galectin-1+OVA treatment group, decreasing to a level comparable to the control group. ELISA results showed that the serum levels of allergen-specific IgE [(0.24 ± 0.06) pg/ml], IL-4 [(104.49 ± 20.76) pg/ml], IL-5 [(82.82 ± 7.71) pg/ml] and IL-13 [(31.59 ± 9.78) pg/ml] were all significantly reduced compared with those in the OVA group [(0.87 ± 0.10) pg/ml, (442.72 ± 14.97) pg/ml, (445.18 ± 35.60) pg/ml, and (434.67 ± 9.78) pg/ml] (P < 0.05). The protein levels of IFN-γ [(120.80 ± 9.71) pg/ml], IL-10 [(63.05 ± 6.05) pg/ml] and TGF-β [(67.89 ± 6.64) pg/ml] in the galectin-1+OVA group were significantly higher than those in the OVA model group [(47.28 ± 5.01) pg/ml, (63.05 ± 6.05) pg/ml and (15.49 ± 3.75) pg/ml] (P < 0.05). The result of flow cytometry examination indicated that the percentage of Treg cells in the galectin-1+OVA treatment group was (9.64 ± 0.41)%, which was significantly higher than (1.81 ± 0.48)% in the OVA model group. Conclusion Galectin-1 inhibits Th2 inflammatory responses in mice with allergic asthma by promoting productions of Treg cells and regulatory cytokines IFN-γ, IL-10 and TGF-β.

The developmental characteristics of Triatoma rubrofasciata under experimental conditions

Li-zhen XIAO, Han-guo XIE, Zhu-yun CHEN, Rong OUYANG, Dian-wei JIANG

2020, 38(2):  219-223.  doi:10.12140/j.issn.1000-7423.2020.02.014

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Objective To investigate the developmental course of Triatoma rubrofasciata, in order to provide referential basis for its studies and control.Methods T. rubrofasciata were collected from Huping village of Hua’an county of Zhangzhou City, Fujiang Province, and maintained at laboratory conditions at 26℃ with relative humidity of 60%, during May 2018 to September 2019. With ICR mice as the only blood supplier(blood supplied for 3 days), the T. rubrofasciata were observed at various developmental stages on hatching, blood sucking, and molting.Results Under the designed experimental conditions, the T. rubrofasciata could completed the whole life history, including 7 stages covering egg stage, nymph stages Ⅰ-Ⅴ and adult stage, lasting for 117.7 days on average. The hatching rate of eggs was 92.2% (391/424). Both nymphs and adults suck blood, nymphs at all stages could not molt until they had sucked enough amount of blood, and female adults could not produce eggs unless they had suck blood. The blood-taking rate and molting rate of stage Ⅰ-Ⅴ nymphs were 86.4% (338/391) and 72.4% (283/391), 76.7% (217/283) and 58.3% (165/283), 71.5% (118/165) and 52.1% (86/165), 91.9% (79/86) and 64.0% (55/86), 96.4% (53/55) and 56.4% (31/55), respectively. The amount of blood taken by the nymphs increased with the growing stages, being (2.9 ± 11.0), (8.0 ± 3.0), (14.9 ± 8.8), (85.4 ± 17.8), and (101.6 ± 54.2) mg for stageⅠ-Ⅴ nymphs, and the folds of body weight gain after blood-feeding were 5.4 ± 1.8, 3.8 ± 1.2, 2.8 ± 1.1, 5.1 ± 1.4, and 2.6 ± 1.1, respectively. The average amount of blood taken was highest at stage V nymphs, and the fold of body weight gain was highest at stage I nymphs.Conclusion T. rubrofasciata can complete its life cycle by feeding animal blood under the experimental conditions, which provides an laboratory platform for related research.

INFORMATION EXCHANGES

Current status of institutional capabilities of officially accredited and approved laboratories in detecting parasites-parasitic diseases in China

Li JIANG, Pu-yan HUANG, Huan-yu Wu, Zhen-yu WANG, Bin ZHENG, Chang-yi GUO

2020, 38(2):  224-233.  doi:10.12140/j.issn.1000-7423.2020.02.015

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Based on the information of officially qualified laboratories undertaking examination and detection service, the current status of operational capacity in detecting parasites-parasitic diseases was descriptively analyzed using the Excel PivotTable tool. The information analyzed were obtained from relevant official websites of government administrations, comprising institution accreditation, laboratory approval, test items, test method, and test standard. The results showed that by the end of June 2019, there were 3 457 testing laboratories at all levels of disease control and prevention centers in China, and 82 (2.4%) of them have been qualified by both China Inspection Body and Laboratory Mandatory Approval (CMA) and China National Accreditation Service for Conformity Assessment (CNAS). 47 of 82 certification and accreditation laboratories detect the parasites (parasitical diseases), accounting for 57.3%. A total of 607 parasitical detection items/parameters were approved (7.4 items/laboratory on average). With regard to the type of testing object, there were 436 items concerning disease control (76.3%), 51 for tap water testing (8.4%), 47 for food inspection (7.7%) and 46 for experimental animal testing (7.6%). Only seven laboratories (14.9%, 7/47) were capable of testing specimens for disease control, as well as tap water and food specimens, which are directly related to human parasitic diseases. With regard to the testing method, there were 403 items (66.4%) using pathogenic methods, 178 items (29.3%) using immunological methods, and 26 items (4.3%) using molecular biological methods. Eleven laboratories (23.4%, 11/47) were qualified being capable of pathogenic, immunological and molecular testings at the same time. Of 607 test items, 75 reference standards were in current use; 437 items (72.0%) using national and trade standards, and 170 items (28%) using non-standard references. Of the 170 items with non-standard references, 89 items (52.4%) have the reference standards available but not befing used. These results suggested that the number of CMA accredited and CNAS qualified laboratories in the disease prevention and control system is low, the average number of testing items per laboratory is not sufficient to cover common parasites, and a considerable number of testing items have not used the standard references, although these standards are already available. To CMA-CNAS laboratories, it is necessary to strengthen the guidance on the development and capacity building, training and capacity validation, in order to further enhance the capabilities of detecting parasite (parasitic disease).

Exploration of multi-form teaching methods in human parasitology education

Xi MA, Xiao-jing SUN, Jia YAO, Wen XU, Wei LI, Li-jun LIU, Yang WANG

2020, 38(2):  234-237.  doi:10.12140/j.issn.1000-7423.2020.02.016

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To improve the teaching quality of human parasitology in accordance to the endemic trend and the demands of clinical practice, we optimized the theory courses of parasitology in teaching content and pattern. A total of 348 undergraduates of two 2016-classes majoring in clinical medicine in Xi’an Medical University were divided into two groups: teaching reform group(176) and control group(172). We explored multi-form teaching approaches applicable for the classes of bigger size based on emerging teaching modes. To the reform group, the reform includes video microlecture, flipped classroom scheduling, introduction of internet resources discussion, and analysis of hot current events and literatures; in control group, the teaching was conducted with routine curriculum. The effect of teaching reform was evaluated in four aspects: classroom outcome, class performance of students, students’ assessment and questionnaire survey. The results showed that the rate of full marks in classroom quizzes was higher in the teaching reform group (49.4%) than that in the control group (27.9%). The average score in final exam in the teaching reform group (81.1 ± 8.4) was significantly higher than that in the control group (73.1 ± 9.0), while there was no significant difference in the passing rate between the two groups (97.7% and 95.9%, respectively). However, the excellent rate in the teaching reform group was significantly higher than that in the control group (10.8% and 2.9%, respectively). The comprehensive evaluation of teachers by students in the teaching reform group was 97.5, higher than 94.4 in the control group(P < 0.05). The recovery rate of questionnaire survey was 92% (162/176). Among the respondents, 90.2% preferred the multi-form teaching method, 57.4% thought highly of the multi-form teaching method in studying parasitology, and 36.4% of students thought this mode had some value. Moreover, the multi-form teaching method resulted in improving study interest(43.2%), cultivating clinical thinking (35.8%) and promoting teacher-student relationships (19.1%). The results showed that, compared to traditional teaching, the multi-mode teaching method can improve students’ interest and autonomous learning of parasitology, improve their academic performance, broaden their knowledge horizon, and enhance their ability to analyze and solve problems.

REVIEWS

Ecological niche modeling and its applications in research on transmission risks of parasitic diseases

Xiao-kang HU, Shang XIA, Yun-hai GUO, Yu-wan HAO, Jing-bo XUE, Shan LV, Jing XU, Shi-zhu LI

2020, 38(2):  238-244.  doi:10.12140/j.issn.1000-7423.2020.02.017

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Ecological niche modeling is developed based on geographic distribution data and associated environmental variables of the species in concern by applying algorithmic approaches to analyze the niche requirements of the species, for predicting the actual and potential distributions of a particular species under different spatiotemporal conditions. Ecological niche modeling is becoming more and more popular in research predicting the transmission risks of infectious diseases. However, the accurate and rational use of the niche modeling relies on fully understanding of the basic principles and characteristics of the modeling. From the view point of studying risks of parasitic diseases transmission, and the concept of ecological niche, this review introduces essential aspects of the ecological niche modeling including the theoretical basis, data collection and variables selection, modeling algorithm, approaches to evaluate the modeling property, and its applications in predicting transmission risks of parasitic diseases.

Advances in diagnostics and treatment of visceral leishmaniasis co-infected with HIV

Xiao-qing HE, Min LIU, Lu ZHOU, Yao-kai CHEN

2020, 38(2):  245-249.  doi:10.12140/j.issn.1000-7423.2020.02.018

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Visceral leishmaniasis is one of the most serious neglected tropical diseases in the world. The coinfection of Leishmania protozoa with human immunodeficiency virus (HIV) has emerged as a serious threat in endemic areas. Due to the interaction between Leishmania parasite and HIV, the coinfected patients display some special clinical manifestations, different diagnostic signs and treatment effects that cause higher mortality and recurrence rate than patients without HIV coinfection. The article aims to review the advances in the clinical manifestation, diagnostics and treatment of visceral leishmaniasis coinfected with HIV.

Advances in the pathogenesis of cholangiocarcinoma caused by Clonorchis sinensis

Xiao LI, Jiang-chang WANG, Lei GUAN, Qi GU, Ji-ze DONG, Chen-yun WU, Min YAN, Zhao-jun WANG

2020, 38(2):  250-254.  doi:10.12140/j.issn.1000-7423.2020.02.019

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Clonorchis sinensis is a food-borne parasite that can cause hepatobiliary lesions and even cholangiocarcinoma. This review summarizes recent progress in the pathogenesis of cholangiocarcinoma caused by C. sinensis, from perspectives of toxicity of worms, abnormal immune responses of hosts and co-infection with other pathogens.

SHORT COMMUNICATIONS

Status of Toxoplasma infection in pregnant and postpartum women in Duyun district of Guizhou Province during 2015-2018

Xiao-qing XU, De-jun LIAO, Xia FENG, Ru-feng XIAO, Jian JIANG

2020, 38(2):  165-167.  doi:10.12140/j.issn.1000-7423.2020.02.020

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To investigate the status of Toxoplasma infection in pregnant and postpartum women in Duyun district of Guizhou Province, China, serum samples were collected during 2015-2018 from pregnant and postpartum women receiving regular checkups and the women who had pathological pregnancies such as miscarriage, stillbirth, and deformity in Qiannan People′s Hospital, to test for specific IgM and IgG antibodies against Toxoplasma by ELISA. SPSS 17.0 software was used for statistical analysis. The positive rates of serum Toxoplasma antibody were compared by chi-square test. A total of 6 931 pregnant and postpartum women participated in the survey, including 6 825 normal pregnancies and parturients, and 106 pathological pregnancies and parturients. The age of the participants ranged 18-45 years, with ethnicities mainly of Han, Buyi, and Miao. The overall positive rate of Toxoplasma antibody was 6.1%(424/6 931), composed of 5.9%(403/6 825) in normal pregnant and postpartum women, and 19.8% (21/106) in pathological pregnancies and parturients (P < 0.01). There was a significant difference in the positive rate of Toxoplasma antibody among Han (7.5%, 215/2 886), Miao (5.4%, 132/2 453), Buyi (5.5%, 71/1 300), Shui (2.3%, 3/131), Tujia (2.0%, 2/100) and Dong (1.6%, 1/61) ethnicities (P < 0.05). There was also a significant difference in the positive rate of Toxoplasma antibody among cadres (1.3%, 22/1 714), teachers (1.8%, 22/1 216), rural workers (13.2%, 320/2 418) and other occupations (3.8%, 60/1 583) (P < 0.01). It was noted that the pregnant women with and without history of contacting with animals was 26.2% (361/1 379) and 1.1% (63/552), respectively, with the difference being statistically significant(P < 0.01). The results demonstrate the pregnant women with positive Toxoplasma antibody in Duyun District of Guizhou Province are mainly those having pathological pregnancyies, being Han ethnicity, engaging in rural farming work, and having contacts with animals.

Affects of Plasmodium berghei infection in mice on spleen B cells and natural killer cells and their surface molecules

Jie-sen ZHANG, Meng-xin ZHANG, Meng-yi HAN, Bing-xia CHEN, Yong-sen LI, Chen-xi JIN, Yan-wei QI

2020, 38(2):  255-258.  doi:10.12140/j.issn.1000-7423.2020.02.021

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To investigate the affects of Plasmodium berghei infection on spleen B cells and NK cells as well as their surface molecules in mice, 10 female C57BL/6 mice were randomly assigned into control group and infection group, five mice each. The mice in the infection group were infected with P. berghei by injection through the tail vein (10 ⁶ parasites/mouse), while the control group received the same volume of saline. Six days after infection, spleens were collected to prepare single cell suspensions, and using flow cytometry to assay the amount of spleen B cells and NK cells and the expression of their surface molecules CD62L, CXCR3 and CD69. The results showed that the percentage of B cells in the infection group was (79.2 ± 3.6)%, significantly higher than that in the control group [(54.3 ± 4.4)%, P < 0.01]. The amount percentage of spleen NK cells in the infection group was (2.2 ± 0.7)%, significantly lower than that in the normal control group [(4.6 ± 0.8)%, P < 0.01]. The percentage of CD62L expression on B cells in the infection group was (77.7 ± 4.4)%, higher than that in the normal group (72.8 ± 7.1)%, but with no statistical significance (P > 0.05). The percentage of CD62L expression on NK cells in the infection group was (61.9 ± 4.8)%, which was significantly lower than that in the control group [(86.6 ± 4.2)%, P < 0.01]. The percentage of CXCR3 expression on B cells in the infection group was (4.1 ± 0.1)%, which was significantly higher than that in the control group [(3.0 ± 0.2) %, P < 0.01]. The percentage of CXCR3 expression on NK cells was (2.1 ± 0.9)%, which was lower than that of the control group [(2.3 ± 0.4)%], but with no statistical significance (P > 0.05). The percentages of CD69 expression on B cells and NK cells were (54.3 ± 4.7)% and (22.2 ± 1.6)%, respectively, both significantly higher than those in the control group [(1.9 ± 0.4)%, (1.3 ± 0.3)%, P < 0.01]. These results suggest that the mice infected with P. berghei may exert the affect against the infection through increasing B cell recruitment and up-regulating the expression of their surface molecules CXCR3 and CD69, and NK cell surface molecule CD69.