25 June 2022, Volume 17 Issue 3

    

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Original Article
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  Establishment of an absolute quantitative method for special intestinal bacterial flora by TaqMan probe multiplex real-time fluorescent PCR

  WANG Hong1, SHI Meifang2, YANG Hao1, WU Yonglin1, XIAO Zhenming1, Qiao Peng1, QU Yifan1, RONG Xingyu1, ZHAO Chao1，3

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 129-138. https://doi.org/10.3969/j.issn.1673-6184.2022.03.001

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  We established a rapid quantification method for detecting the absolute amount of total bacteria, Bifidobacterium, Enterococcus, and Enterobacteriaceae in gut microbiota. The recombinant plasmids for the target DNA fragments of each target bacterial taxa were constructed by molecular clone technique and used as a standard template for quantitative assays. The results showed the primers and probes were highly specific. The linearity of different bacterial standard curves had a high coefficient of determination (R²≥0.995). The linear range was 2.9×10³-2.9×10¹² copies/μL for all bacteria, 3.1×10²-3.1×10⁹ copies/μL for Bifidobacterium, 5.9×10²-5.9×10⁹ copies/μL for Enterococcus and 6.3×10²-6.3×10⁹ copies/μL for Enterobacteriaceae. The low threshold of copy number was 7.5×10²copies/μL for total bacteria, 46 copies/μL for Bifidobacterium, 37 copies/μL for Enterococcus, 51 copies/μL for Enterobacteriaceae, respectively. Moreover, the detection limit was 10 times higher than that of qPCR dye method. Besides, the coefficient of variation of the reproducibility experiment was below 1.32%，implying a well reproducibility. The method was used to detect clinical fecal samples of different ages, and the relationship between these bacteria account and ageing was analyzed, showing high consistent with those reported in other researches. In conclusion, a clinically applicable method for quantification of microbiota was established, which can detect 3 types of bacteria and total bacteria at the same time. And, it has some positive significance for the study of microbiome.
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  Construction and application of an infectious clone of mouse hepatitis virus A59 strain

  YU Yin, FENG Fei, MA Yanlong, ZHU Yunkai, YE Rong, ZHANG Rong

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 139-147. https://doi.org/10.3969/j.issn.1673-6184.2022.03.002

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  Mouse hepatitis virus (MHV) is a model coronavirus, but the traditional etiological methods are limited to the viruses isolated from nature. The reverse genetic system can rapidly obtain the recombinant virus for research, creating a new method to study the genomic functions of MHV and other coronaviruses. In this study, whole genome of the original MHV A59 strain was divided into six fragments by reverse transcription-polymerase chain reaction (RT-PCR). All fragments were individually cloned into plasmid vectors, then the full-length genomic RNA was obtained by restriction enzyme digestion, in vitro ligation and in vitro transcription. After RNA was electroporated into cells, the recombinant virus was rescued. The results revealed that the replication characteristics of recombinant virus rA59 is similar to the original strain, providing an experimental tool for studying the biological characteristics of the virus. To detect the virus replication level, the recombinant reporter viruses rA59-ZsGreen and rA59-Nluc were obtained by inserting the green fluorescent protein ZsGreen (Zoanthus sp. green fluorescent protein) and luciferase Nluc (NanoLuc luciferase) into the open reading frame 4 (ORF4) genome, respectively. The results indicate that the reporter genes do not compromise viral replication and the reporter viruses can well reflect the viral replication property of their parental recombinant virus. These two viruses were used to verify the antiviral activity of remdesivir, by detecting the fluorescence intensity or luciferase activity after treatment. The results demonstrate that the recombinant reporter viruses rA59-ZsGreen and rA59-Nluc are suitable for high-throughput drug screening and have promising potential for application. In summary, the successful establishment of the 6-plasmid-based reverse genetic system for MHV strain A59 provides a powerful tool for studying the biological characteristics of MHV and testing antiviral drugs.
Review
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  Diagnostic value of Xpert MTB/RIF for tuberculosis in human immunodeficiency virus infection/acquired immunodeficiency syndrome patients

  GUO Jing, CHEN Hong, SONG Wei, QI Tangkai, LIU Li, ZHANG Renfang, WANG Zhenyan, TANG Yang, CHEN Jun, SUN Jianjun, XU Shuibao, YANG Junyang, WANG Jiangrong, SHEN Yinzhong

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 148-155. https://doi.org/10.3969/j.issn.1673-6184.2022.03.003

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  The clinical data of 801 human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) patients with suspected tuberculosis were collected to explore the diagnosis value of Xpert Mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) for tuberculosis in HIV/AIDS patients admitted to the Department of Infection and Immunology of Shanghai Public Health Clinical Center affiliated to Fudan University between January 1, 2018 and December 31, 2020. Among 801 HIV/AIDS patients with suspected tuberculosis, 657 patients received tuberculosis screening tests including Xpert MTB/RIF, peripheral blood tuberculosis T cell spot test (T-SPOT.TB), smear microscopy and BACTEC MGIT 960 liquid culture. When the results of liquid culture and Mycobacterium identification and typing were set as the diagnostic criterion for tuberculosis, the sensitivities of Xpert MTB/RIF, T-SPOT.TB, smear microscopy in tuberculosis diagnosis among HIV/AIDS patients (including pulmonary tuberculosis and extra-pulmonary tuberculosis) were 72.8%, 55.4%, 69.6%, respectively, the specificities were 96.8%, 90.3%, 84.4%, respectively, and Kappa values were 0.719 (P<0.01), 0.430 (P<0.01), 0.424 (P<0.01), respectively. The sensitivity and specificity of Xpert MTB/RIF in the diagnosis of pulmonary tuberculosis were 66.7% and 96.0%, respectively. Among sputum smear-positive and sputum smear-negative tuberculosis patients, the sensitivity of Xpert MTB/RIF was 77.4% and 35.2%, respectively, and the specificity was 87.7% and 97.8%, respectively. The sensitivity and specificity of Xpert MTB/RIF in the diagnosis of extra-pulmonary tuberculosis were 63.3% and 95.2%, respectively. The results indicate that Xpert MTB/RIF has high sensitivity and specificity in the diagnosis of tuberculosis in HIV/AIDS patients, and the sensitivity in the diagnosis of pulmonary tuberculosis is higher than that of extra-pulmonary tuberculosis. It is recommended that Xpert MTB/RIF should be used as the preferred testing technique for suspected tuberculosis in HIV/AIDS patients.
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  Yellow fever: current situation and research progress in vaccine and its application

  ZHOU Jiawei1, WANG Jiaqi2, DING Yang1, ZHU Shaoshuai1, FENG Yuqi1, REN Hao2

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 156-162. https://doi.org/10.3969/j.issn.1673-6184.2022.03.004

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  Yellow fever (YF) is an acute hemorrhagic infectious disease transmitted by Aedes mosquitoes, and is endemic mainly in tropical regions such as West Africa and South America. The clinical manifestations of YF include high fever, nausea, vomiting, jaundice, hemorrhage, etc. The etiological pathogen of YF is yellow fever virus (YFV), which is a positive-sense single-stranded RNA virus and belongs to the flavivirus genus of Flaviviridae family. At present, there is no specific antiviral drug for YF, and the general treatment is mainly based on symptomatic and supportive care. Vaccination of an live attenuated yellow fever vaccine (YF-17D) is the most effective way of prevention. Although, YF-17D vaccine has been widely applied and recognized globally, some adverse reactions have been reported occasionally. This review summarizes the epidemic situation, vaccine research progress and application of YFV in current years.
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  Advances in study of efflux pumps of Mycobacterium tuberculosis

  ZHANG Chunjing, XIE Zonghui, WAN Chengjie, YANG Zaichang

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 163-171. https://doi.org/10.3969/j.issn.1673-6184.2022.03.005

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  Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) . With the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), the treatment of TB has become more severe. In recent years, studies have found that the existence of efflux pumps in Mycobacterium tuberculosis is one of the reasons for its drug resistance. The efflux pumps of major facilitator superfamily (MFS), ATP-Binding Cassette (ABC), resistance-nodulation-division (RND) and small multidrug resistance (SMR) have been found in Mtb. However, the drug resistance mediated by Mtb efflux pumps still remains largely unknown, and there is not much work to develop efflux pump inhibitors from the perspective of new drug discovery. This article reviews the structure and function of the efflux pumps of ABC, MFS, RND and SMR of Mtb, as well as the research progress of efflux pump inhibitors of Mtb.
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  Research progress on the correlation between Corynebacterium kroppenstedtii and granulomatous mastitis

  GE Yanmei, TONG Huacheng

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 172-177. https://doi.org/10.3969/j.issn.1673-6184.2022.03.006

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  Corynebacterium kroppenstedtii (C. kroppenstedtii) is a member of the genus Corynebacterium. Genome sequence analysis of the C. kroppenstedtii type strain has revealed a lipophilic (lipid-requiring) lifestyle. The clinical isolates of C. kroppenstedtii have been obtained almost exclusively from female patients, and mainly from the diseased breast tissue samples of the patients with breast abscess and granulomatous mastitis (GM). However, the role of C. kroppenstedtii in breast pathologies remains unclear. This paper reviews the research progress on the correlation between C. kroppenstedtii and granulomatous mastitis.
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  Progress on the relationship between a next-generation probiotic-Akkermansia muciniphlia and chronic disease

  QIN Zhenzhen, LI Fengxia

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 178-183. https://doi.org/10.3969/j.issn.1673-6184.2022.03.007

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  As the research progresses on intestinal flora, more and more evidences show that the imbalance of intestinal flora is closely related to the occurrence and progression of many chronic diseases. Probiotic treatment has become a worldwide hotspot, and Akkermansia muciniphlia (A. muciniphila), a common colonizer in the mucous layer of human intestines, has gradually been regarded as a promising candidate for the next-generation probiotics. The present paper summarizes the ameliorating effects of A. muciniphila on chronic diseases and the possible mechanisms, providing a new idea for the treatment of diseases.
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  Outbreak and etiology of unexplained hepatitis in children in Britain and other countries

  XIE Youhua1，2, CHEN Jieliang1, WANG Yongxiang1, YUAN Zhenghong1，2

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 184-187. https://doi.org/10.3969/j.issn.1673-6184.2022.03.008

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  On 31 March 2022, Scotland first reported five children with unexplained severe hepatitis. The World Health Organization (WHO) issued a guidance on April 15, defining confirmed cases, possible cases and epidemic-related cases of the unexplained hepatitis affecting children. As of April 21, 169 confirmed cases, whose ages ranged from 1 month to 16 years, had been reported in 12 countries. The clinical manifestation was acute hepatitis with aspartate aminotransferase (AST) or alanine aminotransferase (ALT) above 500 IU/L. Many cases had jaundice, nausea, abdominal pain, fatigue, sleepiness and gastrointestinal symptoms like diarrhea and vomiting. Most cases had no fever. Seventeen cases received liver transplantation and at least one death was reported. Considering the epidemiological and clinical characteristics of the cases, infectious factor(s) is more likely to be the etiology. The laboratory examination results of all cases excluded hepatitis A, B, C, D and E, while suggested adenoviruses may be related to the unexplained hepatitis in children, but other infectious factors or environmental factors cannot be completely ruled out. The present paper introduces the development and possible causes of the emerging unexplained hepatitis in children. China should be prepared to cope with an imported risk of this disease.
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  Epidemiology, prevention and control of monkeypox

  ZHU Yuanfei, QU Di

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 188-197. https://doi.org/10.3969/j.issn.1673-6184.2022.03.009

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  Monkeypox is a zoonosis caused by monkeypox virus, which mainly occurs in Central and West Africa. Monkeypox virus can infect a variety of mammals and mainly prevails in the animals. Human can be infected by contacting the virus-infected animals. The clinical manifestations of monkeypox are similar to that of smallpox, but the symptoms are mild. Smallpox vaccination can provide immune protection against monkeypox. However, due to the elimination of smallpox in the world, smallpox vaccination was stopped, and monkeypox has become the most serious orthopoxvirus disease. Recently, the epidemic trend of monkeypox has switched from sporadic cases in natural foci (tropical rain forests in Central and Western Africa) to spread in Europe. On May 7, 2022, monkeypox cases were reported in the UK. Subsequently, more than 100 monkeypox cases and suspected cases were confirmed in Europe. The main transmission ways are contacting with infected animals, and contacting directly or indirectly with patients. On May 20, 2022, the World Health Organization (WHO) held an emergency meeting on the monkeypox epidemic to alert for awareness of monkeypox and to prepare for prevention and response. WHO, the Centers for Disease Control and Prevention (CDC) of the United States and the UK Department of Health reported the relevant monkeypox epidemic and formulated the corresponding prevention and control measures. So far, there is no report of imported monkeypox virus in China. But due to frequent international exchanges, we have to be vigilant for monkeypox. Here this paper will introduce the epidemic status of monkeypox and the information about its prevention and control.
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  Acute severe hepatitis of unknown etiology in children: update of available data and understanding

  ZHANG Li1, XIE Li1, LI Xingyue1, DING Jia2, DENG Qiang1，3, WU Jian1，4，5

  JOURNAL OF MICROBES AND INFECTIONS. 2022, 17(3): 198-208. https://doi.org/10.3969/j.issn.1673-6184.2022.03.010

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  Recently, severe acute hepatitis with unknown etiology in children (SAHUEC) was emerging around the world. Since the first case was found in Scotland, at least 650 cases have been reported in 33 countries. The cases were sporadic, and few appeared to have an epidemiological link. Most children presented as acute hepatitis. In concerning an epidemiological pattern and clinical symptoms, the disease has been considered as an infection with unknown pathogen. Hepatitis A, B, C, D and E were excluded in all cases. Laboratory tests found that more than half of the children were positive for human adenovirus (HAdV), and HAdV-41 was identified in a portion of the cases, suggesting that there was an etiologic link between HAdV infection and SAHUEC. Currently two hypotheses are convincing, that are the adenovirus gene recombination and the altered viral tropism induced by cofactors. Particularly, a superantigen theory is more appealing, i．e. gastrointestinal adenovirus infection induces an onset of SARS-CoV-2 superantigen-mediated toxic shock and hepatocellular apoptosis. However, the true etiology still needs to be determined. This review intends to update available information regarding epidemiological investigation, etiological analysis, pathological alteration, laboratory examination, pathogenic detection, guidelines of diagnosis and treatment, as well as quarantine and preventive measures of SAHUEC, and to provide contemporary understanding of the underlying mechanisms for better therapy.