微生物与感染
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2021 Vol.16 No.2
Published 2021-04-25

Original Article
Review
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79
Establishment of a recombinant hepatitis B virus replicon expressing a split green fluorescent protein
Abstract:Sodium taurcholate cotransport peptide (NTCP) has been identified as a functional receptor with high affinity for hepatitis B virus (HBV). Nevertheless, even in NTCP-reconstituted cells, the low-efficiency of in vitro HBV infection and spread remains a key challenge for the study of the post-entry events in HBV life cycle. In the present study, we developed a recombinant (r) HBV replicon in order to visualize virus-infected living cells via reporter gene expression. We first constructed an HBV replicon vector, i.e. HBV1.1-ΔHBc113, with partial deletion of the sequence encoding HBV core (HBc). The vector demonstrated a competent viral DNA replication in transfected cells, only if HBc was complemented in tans. However, the replication efficiency of ΔHBc113 vector was greatly impaired by insertion of full-length reporter genes being tested. Taking advantage of the property of intein-mediated protein splicing, we chose enhanced-GFP (EGFP) and super folder GFP (sfGFP) as exteins, and developed EGFPN1-8/EGFPC9-11 and sfGFPN1-10/sfGFPC11 split system, respectively. We further constructed EGFPC9-11 or sfGFPC11 recombinant HBV replicons based on the ΔHBc113 vector, the latter of which demonstrated an HBc-rescued, functional intracellular replication, and produced progeny virions in the culture medium. In cells co-expressing EGFPN or sfGFPN, rHBV replicon-expressed EGFPC9-11 or sfGFPC11 were capable of working with their counterparts, respectively, forming intact and functional GFP through intein-mediated protein splicing. We thus successfully developed a fluorescent rHBV replicon system, which can be not only used for cloning HBV-susceptible cell lines, but also have an extensive application prospect for high-throughput antiviral screening.
2021 Vol. 16 (2): 79-87 [Abstract] ( 42 ) [HTML 1KB] [PDF 7754KB] ( 39 )
88
Drug resistance of Helicobacter pylori among children in Shanghai from 2019 to 2020
Abstract: Objective To analyze the resistance of Helicobacter pylori (H. pylori) in children in Shanghai from 2019 to 2020 to four commonly used antibacterial drugs, and provide a basis for the clinical eradication of H. pylori and rational use of antibacterial drugs. Method From January 2019 to October 2020, 1605 children who came to the Children's Hospital of Fudan University in Shanghai and received gastroscopy and rapid urease test were collected. Gastric mucosal membrane samples were collected for H. pylori isolation, culture and identification. E -test method was used for in vitro susceptibility test. Children were divided into 4 groups according to gender and age [1-3 years old (preschool stage), 4-6 years old (kindergarten stage), 7-12 (primary school stage), 13-17 (middle school stage)]. We analyzed the isolation rate of H. pylori, the overall drug resistance rate to metronidazole, clarithromycin, levofloxacin, and amoxicillin and the double/multi-drug resistance. Results A total of 506 H. pylori strains were isolated from the samples of 1605 children enrolled in this study, the isolation rate was 31.6%. There was no gender difference in H. pylori isolation rate (χ2=0.00, P=0.99) and no age difference (χ2=3.945)(P=0.267). The resistance rates of the isolated H. pylori clinical strains to metronidazole, clarithromycin, levofloxacin, and amoxicillin were 42.3%, 25.1%, 8.1%, and 3%, respectively. H. pylori isolated from male and female children were resistant to 4 drugs There was no statistical difference in the rate (χ2=1.042, p=0.307; χ2=0.04, p=0.841; χ2=0.411, p=0.521; χ2=0.623, p=0.430). There was no significant difference in the resistance rates of H. pylori to amoxicillin, levofloxacin and metronidazole (respectively χ2=2.506, P=0.474; χ2= 1.532, P= 0.675; χ2= 2.854, P= 0.46), The resistance rate of H. pylori to clarithromycin in children aged 13-16 years (31.2%) was significantly higher than that in children aged 1 - 3 years (13.3%) (P=0.040). In addition, the resistance rate of clarithromycin + metronidazole was 18%, which was significantly higher than that of clarithromycin + levofloxacin (5.1%), metronidazole + levofloxacin (4.9%), metronidazole + amoxicillin (1.4%) ), amoxicillin + levofloxacin (0.8%) (χ2=172.706, P<0.01). Multi-drug resistance analysis showed that resistance rate of Levofloxacin + clarithromycin + metronidazole was 3.0% , significantly higher than levofloxacin + amoxicillin + clarithromycin (0.6%), amoxicillin + metronidazole + levofloxacin (0.6%) (Χ2=13.907, P=0.01), the H. pylori isolated from another 2 cases were fully resistant to 4 kinds of antibacterial drugs. Conclusion The H. pylori isolated from children in Shanghai from 2019 to 2020 is highly resistant to metronidazole and clarithromycin, and has a low resistance to amoxicillin and levofloxacin. The dual resistance to metronidazole and clarithromycin is relatively high.
2021 Vol. 16 (2): 88-93 [Abstract] ( 22 ) [HTML 1KB] [PDF 533KB] ( 95 )
94
Study on the expressive characteristic of interferon stimulated gene 15 mRNA from peripheral blood of patients with viral pneumonia
This study analyzed the peripheral blood ISG15mRNA expression level in viral pneumonia patients and bacterial pneumonia patients, and discussed the possible relationship between ISG15 and the pathogenesis of viral pneumonia, providing further clinical evidences for host markers of specific respiratory virus infection. 157 adult community acquired pneumonia (CAP) hospitalized patients’ peripheral blood samples and 40 healthy people’s (healthy controls) peripheral blood samples and their clinical data were collected in this study, including 133 patients with respiratory tract infection and 24 patients with bacterial infection. The ISG15mRNA expression level for all samples were analyzed by Real-time quantitative PCR. We validated the ISG15 mRNA expression by stimulating the PBMC in vitro and analyzing the correlation between viral load and ISG15mRNA levels. The expression level of ISG15 mRNA in the respiratory viral infection group was significantly higher than that in the healthy control group and the bacterial infection group. Receiver operating characteristic (ROC) curves analysis showed that the ISG15 mRNA in peripheral blood had a great potential diagnostic value for respiratory virus infection and bacterial infection in adult CAP patients. In vitro studies showed that there was a positive correlation between ISG15mRNA expression in PBMC and viral replication. The peripheral blood ISG15 mRNA expression level were highly correlated with respiratory virus infection by analyzing the clinical data and in vitro experiments. This study demonstrated that ISG15mRNA in peripheral blood may have an important clinical value in diagnosis of viral pneumonia.
2021 Vol. 16 (2): 94-102 [Abstract] ( 22 ) [HTML 1KB] [PDF 1115KB] ( 50 )
103
Isolation and drug resistance of Mycobacterium from clinica specimens of acquired immunodeficiency syndrome patients in Guangxi
To investigate the positive rate, distribution and drug resistance of mycobacteria in clinical specimens of AIDS patients, to provide with reference for clinical diagnosis and treatment. Between January 2010 and December 2019, the clinical specimens of AIDS patients in a hospital in Guangxi were cultured with acid-fast bacilli, and the drug resistance test was conducted with more than 8 anti-tuberculosis drugs after isolation and identification. The overall positive rate of acid-fast bacilli culture was 15.68% (2163/13795),of which the positive rate was high with pus, secretions, various tissue specimens, pleural effusion, and ascites. After species identification, mycobacteria tuberculosis compound group accounted for 77.95% (1442/1850), non-tuberculosis mycobacterium (NTM) accounted for 22.05% (408/1850). The latter accounted for 15.71% - 26.07% over 10 years, with no statistically significant difference between years (χ2=10.442,p>0.05). The total drug resistance rate of acid-fast bacilli was 23.30% (336/1442), the difference over a 10-year period is statistically significant (χ2=18.901, p= 0.026), of which the rate of resistance to isoniazid, rifampicin, streptomycin, ofloxacin, ethambutol, p-aminosalicylic acid, kanamycin was 12.14% (175/336), 10.54% (152/336), 9.29% (134/336), 5.62% (81/336), 3.05% (44/336), 1.80% (26/336), 1.32% (19/336), respectively. The prevalence of MDR-TB, XDR-TB was 5.48% (79/336), 0.28% (4/336), respectively. The study confirmed that the positive rate of mycobacteria isolated from clinical specimens of HIV / AIDS patients is high, of which NTM accounts for a high proportion, and the drug resistance rate of Mycobacterium tuberculosis to commonly used anti-tuberculosis drugs is high. It is suggested that clinical Mycobacterium identification and drug resistance test should be carried out to provide a reliable basis for clinical diagnosis and treatment.
2021 Vol. 16 (2): 103-109 [Abstract] ( 19 ) [HTML 1KB] [PDF 781KB] ( 62 )
 
Review
110 Yi-Kai LU Shu-Ying YUAN Yuan-Bo Yang Qi-Le WU Zi-Xuan FANG Liu-Yan LIU Li CHEN
Research progress on Elizabethkingia meningoseptica and infection
Elizabethkingia meningoseptica (EM), a non-fermented gram-negative bacterium, was one of the most important pathogens of nosocomial infection. The detection rate has been on the rise in recent years. The susceptible people mainly include patients with basic diseases, immunodeficiency or deficiency, long-term use of antibiotics, invasive procedures and so on. EM could cause meningitis, bacteremia, lung infections, eye infections etc. Its multi-drug resistance burdened the clinical treatment. Animals were also infected by EM, like frog, fish, turtle, pig, dog and other infection. The biological characteristics, infection types, detection methods and risk factors were analyzed, which would offer a new insight of EM. Hospitals should focus on detection, drug resistance and pathogenesis of EM, which would provide the reference for prevention and control of nosocomial infection.
2021 Vol. 16 (2): 110-116 [Abstract] ( 16 ) [HTML 1KB] [PDF 943KB] ( 68 )
117
Application Progress of metagenomics technology based on next-generation sequencing platform in the diagnosis of infectious respiratory diseases pathogens
The incidence of respiratory infections is high, early identification of infectious pathogens is the key to improve the cure rate, reduce the mortality.At present, pathogen culture is still the main method of clinical etiology diagnosis, but the low sensitivity and long time are not conducive to early diagnosis and treatment. Metagenomics next-generation sequencing covers the advantages of wide, rapid, unbiased and no specific amplification of pathogens, and has high clinical application value in the identification of rare, mixed infection, immunosuppressive patient infection and the diagnosis of pathogens that are difficult to be detected by conventional detection methods. However, some of them have low specificity, lack of recognized interpretation standards, unclear relationship between sequencing results and treatment, difficulty in testing drug-resistant genes, and high price. At present, in clinical application, metagenomics next-generation sequencing and traditional microbial detection technology have a complementary effect, and the combination of the two can improve the clinical diagnosis efficiency.
2021 Vol. 16 (2): 117-122 [Abstract] ( 18 ) [HTML 1KB] [PDF 638KB] ( 56 )
123
Research Progress in Mechanism of Fusobacterium nucleatum Promoting Colorectal Cancer
Fusobacterium nucleatum is a kind of symbiotic bacteria in human, especially enriched in the oral cavity, and could lead to opportunistic infections in certain cases. Recently, the relationship between Fusobacterium nucleatum and colorectal cancer (CRC) has become a research focus. Numerous clinical studies have shown that Fusobacterium nucleatum is more abundant in CRC, and further studies on animals have proved that Fusobacterium nucleatum can promote the onset and progression of CRC, and chemoresistance to CRC through various mechanisms. We summarized the evidence of the association between Fusobacterium nucleatum and CRC, and provided a comprehensive review of the currently existing mechanisms of Fusobacterium nucleatum promoting CRC.
2021 Vol. 16 (2): 123-128 [Abstract] ( 16 ) [HTML 1KB] [PDF 1031KB] ( 48 )
129
No-antibiotic therapies of Pseudomonas aeruginosa infection
Pseudomonas aeruginosa is the ubiquitous human pathogen causing different infections. The antibiotic therapy is mainly used to deal with the infections caused by P. aeruginosa. The empiric antibiotic therapy ofen does not work due to its resistance to many antibiotics. The non-antibitocs therapies of P. aeruginosa infections are paid extensive attention and many hopeful research progresses are done. This review will focus on the new progresses on the non-antibitocs therapies (including antigen-antibody immunotherapy、phage therapy and antivirulence therapy ) of P. aeruginosa infections, in addition, the problems touse these new therapies in the clinical treatment and the possible strategies to overcome them are also discussed.
2021 Vol. 16 (2): 129-136 [Abstract] ( 14 ) [HTML 1KB] [PDF 875KB] ( 64 )
137
Research progress on vertical transmission of Chikungunya virus
Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes aegypti and Aedes albopictus, and belongs to the Alphavirus genus of Togaviridae family. Since 2005, several epidemics of CHIKV with high infection rate have been reported in Africa, Asia, and the Americas. The infection of CHIKV causes fever and arthritis which usually lasts for months or even years, resulting in a big burden to patients and society. Besides mosquito-borne transmission, vertical transmission from pregnant women infected with CHIKV during pregnancy to fetus or neonatal were occasionally reported, which may lead to severe neurological sequelae of the newborn. Here, we will summarize the vertical transmission cases of CHIKV reported so far, review the relevant clinical manifestations, propose underlying modes and mechanisms, analyses the advantages and disadvantages of the present animal model used in the field, and discuss the diagnosis, prevention and treatment of this disease, in order to provide fundamental information for in-depth study of vertical transmission of CHIKV.
2021 Vol. 16 (2): 137-144 [Abstract] ( 17 ) [HTML 1KB] [PDF 3014KB] ( 40 )
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