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Development of an immunochromatographic strip for detection of Zika virus NS1 antigen

Sui⁃bin HUANG ; Ling⁃hong XIONG ; YANG Fan ; Xiao⁃min ZHANG

Journal of Tropical Medicine.2019;19(11):1327-1331.

Abstract：Objective To develop a colloidal gold immunochromatographic strip ⁃ based method for the rapid detection of Zika virus（ZIKV）NS1 antigen. Methods The gold nanoparticles modified with the anti⁃ZIKV NS1 monoclonal antibody as the detection probe were coated on the glass ⁃fiber pad. The anti ⁃ZIKV NS1 monoclonal antibody and the goat anti ⁃mouse polyclonal antibody were immobilized on a nitrocellulose membrane as the test line and the control line，respectively. In order to achieve critical results，the ratio of the optical density （OD）of the test line to that of the control line was compared. Serial diluted ZIKV NS1 standard antigen was applied to evaluate sensitivity of the immunoassay. The culture supernatant and serum samples for arboviruses（ZIKV，Dengue virus, Japanese encephalitis virus and Chikungunya virus） were utilized to demonstrate the specificity of the method. Results The detection result could read by naked eyes within 20 minutes. The visual cut ⁃off level for the test strip was achieved at 100 ng/mL of the Zika virus NS1 standard antigen. No cross⁃reactions with Dengue virus，Japanese encephalitis virus and Chikungunya virus were observed. The strip could remain good stability within 36 weeks whether stored in 4 ℃ or room temperature（22-25 ℃）. Conclusion Apart from stability， the method was convenient，rapid and specific for ZIKV NS1 antigen，which showed a promising potential in the point of care test and the screening test.

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A new method for rapid detection of dengue antigen based on microfluidic immune magnetic beads

Su⁃xiang LIN

Journal of Tropical Medicine.2019;19(11):1336-.

Abstract：Objective To establish a sensitive，rapid and convenient method for the detection of dengue antigen and assist clinical diagnosis of dengue. Methods In this paper，we developed a rapid detection method for dengue antigen based on microfluidic immune magnetic beads. Solidwork software was used to design microfluidic chip，which was prepared by mechanical processing and chemical sealing. Immunomagnetic beads of dengue antibody were prepared by chemical coupling reaction. Using HRP ⁃TMB ⁃H2O2 as color system，dengue NS1 antigen was detected on microfluidic chip carrier by double antibody sandwich method. Finally，57 clinical samples were tested by the novel method and traditional ELISA kit，and the accuracy of the method was analyzed，and the advantages and disadvantages of the two methods were compared. Results 20 minutes was needed to detect dengue NS1 antigen by using the novel ELISA method，and the reaction system only needed 10 μg beads and 10 μL samples. In the verification experiment，the method could distinguish the negative from the positive obviously. The positive sample had color rendering，while the negative and blank samples had no color rendering. In terms of detection performance，the coincidence rate between the new ELISA method and the traditional ELISA method reached 100%. Conclusion The novel ELISA detection platform had the advantages of simple，rapid，reagent and sample saving，high sensitivity，good stability and high accuracy，and could be used for the detection of dengue antigen.

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The use of a multiplex real⁃time PCR assay for rapid diagnosing acute respiratory viral infections in children

Jia⁃nan XU

Journal of Tropical Medicine.2019;19(12):1468-.

Respiratory tract infection；Multiplex PCR；Children Abstract:Objective The aim of the study was to evaluate a multiplex molecular real ⁃ time assay for the diagnosis of respiratory viruses of childhood acute respiratory virus infection，and analyze the symptoms associated with the pathogens which were detected from those samples. Methods From August 2018 to August 2019，a sample of 275 swallow swabs from children with acute respiratory infections in Chengdu Children′s Specialist Hospital was collected and tested using the RespiFinder®SMART 22 test kit. The kit is based on multiplex PCR technology and can detect and distinguish 22 pathogens related to respiratory infections at one time. Results Of the 275 samples，245（89.1%）detected positive viral infections， of which 160 were single virus infections，59 were multiple viral infections，17 were positive for Mycoplasma pneumoniae， and 9 were positive for Chlamydia pneumoniae. The most common pathogens were rhinovirus 77 cases（28.0%），adenovirus 45 cases（16.4%）and respiratory syncytial virus 34 cases（12.4%）. Influenza virus was detected in 28（10.2%）samples， parainfluenza in 25 cases（9.1%），coronavirus in 15 cases（5.5%），Boca virus in 12 cases（4.4%），and human metapneumovirus in 9 cases （3.3%）. Rhinovirus （28.0%） was the most common pathogen in upper respiratory tract infections，while respiratory syncytial virus（12.4%）was the most common pathogen in lower respiratory tract infections. It has been found that multiple viral infections in children were associated with severe respiratory symptoms in clinical manifestations. Conclusion Compared with ordinary PCR detection，the multiplex PCR could detect multiple pathogens at one time in experiment，which greatly shortens the detection time and had important clinical significance for rapid diagnosis of multiple respiratory pathogens. Thus，it could be useful for detecting several viruses causing respiratory tract disorders.

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Public practice，attitude and knowledge of coronavirus disease

Ye Qi ; Liu-huan CHEN ; Li ZHANG ; Ying⁃ying YANG ; Si⁃yi ZHAN ; Chuan⁃xi FU

Journal of Tropical Medicine.2020;20(02):145-149.

Objective To analyze the status of knowledge，attitude and practice of the coronavirus disease（COVID ⁃19）among the adults of 18⁃59 years old in China，and to provide scientific basis for corresponding health education strategies. Methods In the rapid development phrase of COVID ⁃ 19，subjects from all provinces or municipalities of China were invited to participate in a quick questionnaire online survey on January 29th，2020. Results The effective response rate of completing questionnaire was 97.41%（3 083/3 165）. 98.54% of the subjects reported that they were very terrified. The main reasons included the high contagion（64.71%）and lack of effective treatments（19.92%）；94.45% of the subjects were concerned that they and their family members would be infected by the novel coronavirus. 99.42% knew that the virus could be transmitted from person to person；97.89% and 93.87% knew that it could spread through respiratory tract and contact respectively，97.73% knew that patients without symptom could also be contagious，96.37% knew that persons in close contact to COVID⁃19 patient were required to be quarantined for at least 14 days of medical observation. 99.09% knew that the pathogen of this disease was novel coronavirus. 65.46% knew that both medical protective masks and surgical masks could prevent COVID⁃19 effectively. 99.68% had confidence in defeating COVID⁃19，and 85.86% believed that COVID⁃19 would be controlled within the next 3 months. Study subjects mainly obtained health information through WeChat（88.97%）or websites（82.06%）. The proportions of the subjects who can cover mouth and nose when coughing or sneezing，avoid hand contacting with eyes，mouth or nose，practice hand hygiene，wear masks outside，avoid exposure to respiratory patients，and avoid the crowded were 89.85%，85.44%，95.13%，96.89%，92.18% and 96.27%，respectively. Multivariate Logistic analysis showed that gender（OR=0.544，95%CI：0.440⁃0.673，P<0.001），age（OR=1.844，95%CI：1.466⁃2.320，P<0.001），recognition （OR=2.200，95% CI：1.780 ⁃ 2.718，P<0.001） were associated with those good behaviors. Conclusion After the happened，the government and society′s vigorous publicity to the public achieved good results. The public are highly concerned and have a high awareness of the knowledge of COVID ⁃ 19. They adopt protective measures proactively. Females，middle⁃aged，and individuals with insufficient recognition are likely under⁃protected. In the different epidemic stages of the emerging infectious disease，health education should be carried out to the public based on scientific evidences.

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Determination of Circulating Antigen in Neurocysticercosis Patients by Monoclonal Antibody

Zengzhu GUO ; Minjun HUANG ; Yijun AN ; Song HUANG ; Hongjie JIANG

Journal of Tropical Medicine.2005;5(2):113-115.

Objective To detect circulating antigen (Cag) for diagnosing neurocysticercosis. Method ELISA was performed with monoclonal antibody 4B6 against the cyst fluid antigen of Cyticercus cellulosae for detecting Cag in serum and/or cerebrospinal fluid (CSF) of patients with neurocysticercosis or with other diseases. Results In the group of 82 cases of neurocysticercosis, the positive rate of serum Cag was 79.2% (65/82) and the positive rate of CSF Cag was 100% (26/26). After chemotherapy for 20 cases with positive serum Cag, the titer of serum Cag in 17 cases dropped to zero(85% ). Cag could not be detected in specimens from patients with other diseases. Conclusion These results indicate that the determination of Cag, especially of the CSF Cag, is useful for the diagnosis of neurocysticercosis and the drop in serum Cag is a good parameter for the evaluation of the effectiveness of chemotherapy.

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Animal Host of Schistosoma japonicum and Transmission of Schistosomiasis in Poyang Lake Region

Dandan LIN ; Yueming LIU ; Fei HU ; Shaoji ZHANG

Journal of Tropical Medicine.2003;3(4):383-387.

The article clarified, according to the recent research results which have been done in Poyang Lake region of Jiangxi province, bovines are the most important animal hosts of Schistosoma japonicum and infected bovines are main infectious sources of Schistosomiasis in Poyang Lake region. Spring (March to June) is the " cross transmission stage" between definitive host and intermediate host, that is, spring is not only the susceptible season of infection for livestock and human, but also is the susceptible season of infection for snails. In flood season (July to August), the transmission of Schistosomiasis between livestock and snails belong to " low level phase" . Autumn (September to Octber) is the second seasonal peak of cercariae and is the susceptible season of infection for livestock and human. Winter(November to Feburary in next year) is the non- susceptible season of infection. Most of the susceptible zones of infection for livestock, human and snails are the marshlands near the endemic villages.

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Primary Study of the Structure and Antigenic Epitopes of Antigen Pf332

Zhixin SHAN ; Xinbing YU ; Jin XU ; Zhongdao WU ; Xuerong LI ; Guowu BIAN ; Changling MA ; Yan LI ; Shouyi CHEN ; Xuchu HU

Journal of Tropical Medicine.2002;2(3):219-224.

Objective To understand the primary structure and potential antigenic epitopes of antigen Pf332(Ag332) of P.falciparum iso late FCC1/HN.Methods　Based on the published Pf332 gene sequence , nine pairs of primers were designed for the PCR amplification of the Pf332 gen e fragments from genomic DNA of P.falciparum isolate FCC1/HN. The amplified gene fragments were subcloned into pMD-18T vectors and sequenced. The sequences were aligned using DNAstar software to obtain the full-length sequence of the gene Pf332. The primary structure and sequence homology of Ag332 were analyzed by SAPS, Tmpred, SingalP and Blastn programs. Three fragments, R0, R1 and R2, cor responding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA3-S separately. The Balb/c mice were immunized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3- S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA I mmunization were analyzed by ELISA and parasite growth inhibition tests in vitro .Results　Nine Pf332 gene fragments were specifically amplif ied, subcloned into pMD-18T vectors and sequenced. Pf332 gene of the P.falci parum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 ami no acids, about 615.28kDa. The Ag332 contains 17 regions of highly degenerated Glu-rich repeats, with 30.18% Glu in total amino acids of Ag332. Ag332 of P.falciparum isolate FCC1/HN and 3D7 exhibited 94.55 % homology in amino acid residues. The results of immunohischemistry assay showed that R0, R1 and R2 were expressed in mice muscle tissue. The amount of IgG antibody of the groups immu nized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 were higher than those of blank and pcDNA3 groups (P＜0.05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 had an incomplete inhibitor y effect on P.falciparum growth. Conclusion　The antigen Pf332 is an large protein containing highly degenerated Glu-rich repeats. Pf332 gene fragments, R1 and R2 encoding potent antigenic epitope repeats.

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Cloning Expression and Characterization of Two Cysteine Proteinases of Clonorchis sinensis

Fuquan PEI ; Nagano ISAO ; Jun WU ; Zhiliang WU ; Huier CUI ; Takahashi YUZO ; Bo PAN ; Yueyi FANG

Journal of Tropical Medicine.2004;4(1):10-14,55.

Objective To clone and express the valuable Clonorchis sinensis antigen molecules which can be applied to the diagnosis of clonorchiasis. Methods Based on the sequences (Genbank) No. AF271091 (CysA) and No.AF093242 (CysB), primers were designed to amplify the two C. sinensis cysteine proteinase genes and expressed in E.cloi. The expressed proteins were purified by affinity chromatography and then tested for their immunological characters.Results The two genes were successfully cloned and expressed. Western blot showed that CysB had strong reaction with clonorchiasis sera and very weak reaction with schistosomiasis sera, while CysA showed no reactivity with the probed sera. Immunohistochemistry showed that both proteins were mainly located in adult worm intestines and the intrauterine eggs.Conclusions The results suggested that, of the two expressed C. sinensis proteins, CysB had good antigenic reactivity against sera from patients. It is a potential candidate of diagnostic antigens for clonorchiasis.

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A Novel Rabl-like Gene with A 25 bp Intron in Trichomonas vaginalis

Lili LUO ; Xiaoyuan XU ; Mingyan XU ; Yucai FU

Journal of Tropical Medicine.2008;8(7):633-638,648.

Objective The aim of this study is to clone and characterize a novel Trichomonas vaginalis Rabl-like geue (TvRabl-like) with a small intron. Methods The eDNA clone of TvRabl-like gene was isolated from a cDNA expression library and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST ClustalW programs.Phylogenetic analysis was carried out by MEGA3 program. The genomic DNA and mRNA of TvRab1-like gene were amplified using PCR and RT-PCR techniques respectively and also sequenced. Results The eDNA sequence of TvRab1-like gene had a length of 705 base pairs with an open reading frame of 603 bp. The deduced amino acid sequence from the open reading frame possessed 200 residuals corresponding to a putative M.W. 22532.2 and an estimated pl of 7.4. Sequence analysis demonstrated that TvRab1-like gene showed the highest homology to T. vaginalis Rabla (63% identity and 79% similarity) and the Rabl subfamily of other species, suggesting that the deduced amino acid sequence from this cDNA clone was a Rabl isoform. Phylogenetic analysis showed that TvRab1-like gene was clustered with T.vaginalis Rab1 subfamily in the phylogenetic tree. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA possessed a 25 bp intron which contained canonical 5' GT-AG-3' and branch site motifs as those larger introns found in T.vaginalis and other eukaryotes. The analysis of RT-PCR products demonstrated the presence of the unspliced mRNA and spliced mRNA, indicating that there was a intron. Conclusion These data suggest that TvRabl-like gene belongs to T.vaginalis Rabl subfamily. TvRabl-like gene possesses a 25 bp splieeosomal intron which is the smallest one of the introns identified in this deepest-branching protist and might be the shortest intron of eukaryotes. Study of those introns might provide more insights into the intron evolution of eukaryotes.

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Induction of Apoptotic-like Cell Death in Trichomonas vaginalis by Metronidazole

Petrus TANG ; Chingcheng HUANG ; Jyhwei SHIN ; Renli ZHANG ; Juli LIU ; Yucai FU

Journal of Tropical Medicine.2007;7(9):837-841,849.

Objective Apoptosis or programmed cell death(PCD) has been studied extensively in multicellular organisms,however,very little is known about the molecular mechanisms by which apoptosis occurs in unicellular protozoan parasites.The aim of this study is to characterize the apoptosis or PCD of Trichomonas vaginalis induced by metronidazole (MTZ).Methods T. Vaginalis strain cultures were treated with various concentrations of MTZ and the number of viable cells were determined at different time intervals.The genomic DNA of MTZ treated T. Vaginalis was extracted and DNA fragmentation was analyzed.TUNEL assay was carried out to detect the endonuclease activity in T. Vaginalis after MTZ treatment.Flow cytometric analysis was used to analyse the phosphatidylserine (PS) exposure of T. Vaginalis.Results Metronidazole (MTZ) induced an apoptotic-like cell death in T. Vaginalis.This apoptotic-like cell death was demonstrated by cell shrinkage,phosphatidylserine exposure,and nuclear chromatin condensation.However, no oligonucleosmal DNA laddering was detected.Conclusion The regulatory pathway of apoptotic cell death in T. Vaginalis may be different from multicellular organisms.The determination of protozoan apoptotic pathways leading to cell death might ultimately allow the identification of new therapeutic targets.

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