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OBJECTIVES@#To explore the value of metagenomic next-generation sequencing (mNGS) in the pathogen identification in children with hematological malignancies complicated with infections.@*METHODS@#A retrospective analysis was conducted on clinical data and pathogenic test results of 43 children with hematological malignancies who underwent microbial culture and mNGS due to infections in the Third Xiangya Hospital of Central South University between June 2020 and July 2022. Differences in detection rates and characteristics of pathogenic microorganisms detected by mNGS and microbial culture were compared.@*RESULTS@#A total of 54 specimens were examined, and the overall detection rate of pathogen by mNGS (80%, 43/54) was significantly higher than that by microbial culture (30%, 16/54) (P<0.001). The most commonly detected infection type by mNGS was viral infection, followed by fungal infection combined viral infection, while that by microbial culture was bacterial infection, followed by fungal infection. The detection rate of fungi by mNGS (33%, 18/54) was higher than that by microbial culture (6%, 3/54) (P<0.001). The detection rate of two or more pathogenic microorganisms by mNGS was higher at 48% compared to microbial culture at 9% (P<0.05). The detection rate of two or more types of pathogenic microorganisms by mNGS was also significantly higher at 33% compared to microbial culture at 2% (P<0.05). The most commonly detected bacteria and fungi by mNGS were Pseudomonas aeruginosa and Candida tropicalis, respectively, in peripheral blood, while Streptococcus pneumoniae and Pneumocystis jirovecii were most commonly detected in bronchoalveolar lavage fluid. Treatment adjustments based on mNGS results were beneficial for 35% (15/43) of the cases.@*CONCLUSIONS@#mNGS has a higher detection rate than microbial culture and has obvious advantages in diagnosing mixed and fungal infections, making it a useful supplementary diagnostic method to microbial culture.
Subject(s)
Humans , Child , Retrospective Studies , Hematologic Neoplasms/complications , High-Throughput Nucleotide Sequencing , Bronchoalveolar Lavage Fluid , Hospitals , Sensitivity and SpecificityABSTRACT
Objective:To explore the value of detecting pneumocystis carini(PC)rapidly in immunocompromised patients by loop mediated isothermal amplification(LAMP).Methods:Respiratory tract specimens of immunocompromised children suspected of pneumocystis carinii pneumonia(PCP) at Shanghai Children′s Medical Center, School of Medicine, Shanghai Jiao Tong University were collected from May 2020 to May 2021.PCR and LAMP methods were used to detect PC.Firstly, LAMP primers of PC were synthetized according to the conserved region of PC gene, and the LAMP reaction system and reaction conditions were optimized to evaluate the sensitivity and specificity.Then, the results of pathogens were compared with those of PCR detection.Results:The established LAMP detection technology for PC had high specificity and super sensitivity.The detection results could be obtained within 1 hour.In 12 clinical samples, 10 cases were positive and 2 cases were negative, the coincidence rate of LAMP and PCR technique was 100%.Conclusion:LAMP can detect PC more rapidly and sensitively than PCR, and it can provide a good support for clinical rapid diagnosis of PCP.
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Objective:To explore the value of detecting pneumocystis carini(PC)rapidly in immunocompromised patients by loop mediated isothermal amplification(LAMP).Methods:Respiratory tract specimens of immunocompromised children suspected of pneumocystis carinii pneumonia(PCP) at Shanghai Children′s Medical Center, School of Medicine, Shanghai Jiao Tong University were collected from May 2020 to May 2021.PCR and LAMP methods were used to detect PC.Firstly, LAMP primers of PC were synthetized according to the conserved region of PC gene, and the LAMP reaction system and reaction conditions were optimized to evaluate the sensitivity and specificity.Then, the results of pathogens were compared with those of PCR detection.Results:The established LAMP detection technology for PC had high specificity and super sensitivity.The detection results could be obtained within 1 hour.In 12 clinical samples, 10 cases were positive and 2 cases were negative, the coincidence rate of LAMP and PCR technique was 100%.Conclusion:LAMP can detect PC more rapidly and sensitively than PCR, and it can provide a good support for clinical rapid diagnosis of PCP.
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Infectious diseases are still the most important diseases in infants and young children.Due to young age and imperfect immune function, infectious diseases in children are characterized by "urgent" , "serious" and "complex pathogens" , which often lead to serious complications.Rapid and accurate diagnosis is particularly important for children.However, the existing pathogen detection methods are inefficiency, subject to experimental conditions and person, and impopularized in hospitals.The pathogen detection method based on loop mediated isothermal amplification presents high specificity, low-cost and simple operation and is suitable for promotion in primary medical units, which has great application value in the detection of pathogens of children.In addition, the combination of loop mediated isothermal with paper-based micro-gene chip, intelligent automation and digital system will make progress to clinical etiological diagnosis.
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Objective:To analyze the distribution and drug sensitivity of pathogens in bronchoalveolar lavage fluid(BALF)of children with severe community acquired pneumonia(CAP)in Qingdao from 2018 to 2020.Methods:The clinical data of 482 children with severe CAP in Qingdao admitted to Women and Children′s Hospital of Qingdao University were collected.BALF was collected by bronchoscopy for detection of bacteria and mycoplasma.Results:(1)Bacterial infection was detected in 139 cases(27.84%), mycoplasma infection in 119 cases(24.69%), and virus infection in 141 cases(29.25%). (2)The detection rates of bacteria and virus infection in the 1-12 months old group were higher.The detection rate of mycoplasma pneumoniae was the highest in the group over 5 years old.(3)A total of 139 strains were positive in bacterial culture of lavage fluid under bronchoscope: 55 strains(39.57%) of gram-negative bacilli and 84 strains(60.43%) of gram-positive cocci.Streptococcus pneumoniae was the most common gram-positive bacteria.Haemophilus influenzae was the most common gram-negative strain.(4)Streptococcus pneumoniae and Staphylococcus aureus were highly sensitive to amoxicillin clavulanate potassium, vancomycin and linezolid.The resistance rate to erythromycin was high(100%). (5)Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae were highly sensitive to meropenem and cefoperazone sulbactam.They were highly resistant to amoxicillin, ampicillin and cefuroxime(>80%).Conclusion:Severe CAP in Qingdao area is mainly caused by virus and bacteria within 1 year old.Mycoplasma pneumoniae infection is the main cause of children over 5 years old.Respiratory syncytial virus, adenovirus and parainfluenza virus are main causes of virus infection.Streptococcus pneumoniae and haemophilus influenzae are the main pathogens, which are more sensitive to vancomycin, linezolid, meropenem and cefoperazone sulbactam, but resistant to erythromycin and amoxicillin.
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Infectious diseases are commonly seen in clinical practice, and pathogen diagnosis is the key link in diagnosis and treatment; however, conventional pathogen detection methods cannot meet clinical needs due to time-consuming operation and low positive rate. As a new pathogen detection method, metagenomic next-generation sequencing (mNGS) has a wide detection range and can detect bacteria, viruses, fungi, parasites, rare pathogens, and even unknown pathogens. The technique of mNGS is unbiased and can rapidly, efficiently, and accurately obtain all nucleic acid information in test samples, analyze pathogens, and guide clinical diagnosis and treatment, thereby playing an important role in complicated infectious diseases. This article reviews the diagnostic advantages and clinical value of mNGS in bacterial, fungal, viral, and parasitic infections.
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Humans , Bacteria , Communicable Diseases/diagnosis , High-Throughput Nucleotide Sequencing/methods , Metagenomics/methods , Sensitivity and SpecificityABSTRACT
The cultivation and production of cucumber are seriously affected by downy mildew caused by Pseudoperonospora cubensis. Downy mildew damages leaves, stems and inflorescences, and then reduces the yield and quality of cucumber. This review summarized the research advances in cucumber downy mildew, including pathogen detection and defense pathways, regulatory factors, mining of pathogens-resistant candidate genes, proteomic and genomic analysis, and development of QTL remarks. This review may facilitate clarifying the resistance mechanisms of cucumber to downy mildew.
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Cucumis sativus/genetics , Oomycetes/genetics , Peronospora , Plant Diseases/genetics , ProteomicsABSTRACT
In the past, coronavirus caused two serious human-to-human pandemics in the world, including severe acute respiratory syndrome （SARS） and Middle East respiratory syndrome （MERS）. In late 2019, coronavirus disease 2019 （COVID-19） caused another major global public health event. Due to the strong infectivity of novel coronavirus, it is difficult to carry out the autopsy of related death cases widely. This paper reviews the previous status of the pathogen detection related to the autopsy of coronavirus infection diseases, and introduces the ongoing detection methods of novel coronavirus in clinical practice, in order to provide reference for the pathogen detection and study related to autopsy of COVID-19.
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Humans , Autopsy , COVID-19 , Communicable Diseases , Coronavirus Infections/diagnosis , Middle East Respiratory Syndrome Coronavirus , SARS-CoV-2ABSTRACT
Next-generation sequencing (NGS), also known as high-throughput sequencing, is more efficient compared with Sanger sequencing that has become the standard method of clinical DNA sequencing, and can obtain a large amount of information in a relatively short time at a lower cost.NGS has broad prospects in such aspects as diagnosing the pathogen of lower respiratory tract infection in children, identifying the pathogen of cross-infection in hospital, drug resistance research and vaccine development.It is still worth conducting further studies on the approach to improve the specificity and sensitivity of diagnosis and to optimize NGS.
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Objective To understand the patterns and epidemiological characteristics of severe pneumonia in Shapingba District, Chongqing area, and to provide reference for prevention and treatment of severe pneumonia. Methods The clinical data of 174 patients with severe pneumonia admitted to our hospital from January 2018 to December 2020 was retrospectively analyzed, including general demographic data, pathogenic test results, deaths, etc., and statistical analysis was performed. Results Fifty-four cases (30.5%) of 174 patients with severe pneumonia were positive for influenza virus, including 28 cases of new A H1N1, 11 cases of human H7N9, 9 cases of seasonal H3, and 6 cases of influenza B.In 174 cases of severe pneumonia patients,there were 105 males (60.34%) and 69 females (39.66%) withthe onset time: 63 cases (36.21%) from March to May, 5 cases (2.87%) from June to August, and 22 cases from September to November (12.64%), 84 cases (48.28%) from December to February.Eighty-two of 174 patients(47.13%) with severe pneumonia died. Logistic regression analysis showed that older age, more than 3 organs involved, COPD, septic shock, and influenza virus infection were risk factors for severe pneumonia death. Conclusion Severe pneumonia in Shapingba, Chongqing area is dominated by influenza A virus infection, which usually occurs in winter and spring with a high mortality rate. It is necessary to conduct disease prevention and control for high-risk groups.
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Objective The present study was conducted to identify the Vibrio cholera type and to analyze its antibiotic resistance in an epidemic of cholera in Haiyan County in 2018, which would provide the references for prevention and control of cholera. Methods Stool samples of the patient and his close contacts as well as the food and environmental samples were collected for identification of the type of Vibrio cholerae and the toxin gene. The resistance of identified Vibrio cholerae to 20 different common antibiotics were tested. Results A total of 176 samples were collected, including 101 stool samples from the case and his close contacts, 35 environmental samples and 40 food samples. Among those samples, only one strain of V. cholerae, O139, was isolated from the patient's first feces sample. It was detected as a toxin gene of ctxA positive by real-time fluorescence PCR. Antibiotic resistance test showed that the strain was sensitive to norfloxacin, levofloxacin, ciprofloxacin, cefotaxime, cephalothin, ampicillin, and amoxicillin. It was 100% resistant to tetracycline, doxycycline, neomycin, kanamycin, streptomycin, and rifampicin. Conclusion V. cholerae O139 strain with ctxA is detected in an epidemic of cholera. Norfloxacin, levofluoxacin and some other antibiotics could be used for clinical treatment and prevention. It should pay attention to this strain of V. cholera regarding the multiple drug resistance and the change of antibiotic resistance.
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The Neon-S method has been used for detection of Sclerotinia sclerotiorum on soybean and common bean seeds since the 2010 crop season. However, this method can lead to identification of false-positives due to the presence of other fungi that change the medium pH. Thus, this study evaluated the effect of increasing incubation period on the reliability of Neon-S test in detecting S. sclerotiorum infection on soybean and common bean seeds. A randomized block design was set up with three replicates in a 3x3 factorial scheme, consisting of three detection methods (germination paper test, Neon-S, and modified Neon-S2) and three seed material (naturally infected common beans, naturally infected and artificially inoculated soybean seeds). The three methods were compared by evaluating 400 seeds per replication, after incubating them for seven days in Neon-S, for 15 days in Neon-S2, and for 30 days in germination paper, determining the presence of the fungus and of sclerotia adhered to the seeds. The data were submitted to the analysis of variance and the averages compared by the Tukey test at 5% probability. From 2008 to 2012, 637 lots were evaluated. Among the seed material, artificially inoculated soybean presented the greatest pathogen infection index. The germination paper test led to 2.8% of positive samples, contrasting 29.7% of Neon-S. The modified method Neon-S2 increased detection sensitivity of S. sclerotiorum in seed lots (31.2%); however, did not significantly differ from the Neon-S method, despite its greater averages. We concluded that detection of S. sclerotiorum by the Neon-S method can be optimized by incubation for 15 days (Neon-S2), due to the formation of sclerotia near the infect seeds which confirms the presence of the pathogen avoiding false-positive results.
O método de Neon-S tem sido utilizado para a detecção de Sclerotinia sclerotiorum em sementes de soja e de feijão desde a safra de 2010. Porém, esse método possibilita a leitura de falsos-positivos devido ao aparecimento de fungos que também alteram o pH do meio. O objetivo deste trabalho foi verificar se o aumento do período de incubação melhora a confiabilidade do teste Neon-S em detectar o patógeno S. sclerotiorum em sementes de soja e de feijão. Utilizou-se o delineamento experimental de blocos casualizados, em esquema fatorial 3x3, sendo três métodos de detecção (rolo de papel, Neon-S e o meio modificado Neon-S2) e três tipos de sementes (feijão infectado naturalmente e sementes de soja infectadas natural ou artificialmente), totalizando nove tratamentos, com três repetições. Os três métodos foram comparados avaliando 400 sementes por repetição: em meio Neon-S com incubação de sete dias, Neon-S2 com incubação de 15 dias e em rolo de papel por 30 dias, anotando-se a presença do fungo e de escleródios aderidos às sementes. Realizou-se a análise de variância dos dados e teste de comparação de médias (Tukey 5%). No período de 2008 a 2012, 637 lotes foram testados. Dentre os tipos de sementes, a soja inoculada artificialmente apresentou os maiores índices de infecção pelo patógeno. O teste de rolo de papel apresentou 2,8% de amostras positivas, enquanto o Neon-S 29,7%. O método Neon-S2 aumentou a sensibilidade de detecção de S. sclerotiorum nos lotes de sementes analisadas (31,2%); porém, não foi detectada diferença significativa comparativamente ao método Neon-S, ainda que com maiores médias. Conlui-se que a detecção de S. sclerotiorum pelo método Neon-S pode ser otimizada com a incubação por 15 dias (Neon-S2), em virtude da formação de escleródios próximos às sementes infectadas, o que confirma a presença do patógeno e evita a leitura de falsos-positivos.
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Ascomycota , Seeds , Soybeans , Phaseolus , Fungi , Noxae , FabaceaeABSTRACT
Objective To investigate the pathogenic characteristics of patients with ventilator associated pneumonia ,and analyze the correlation with the changes of serum procalcitonin (PCT ) ,C reactive protein (CRP) and pulmonary function .Methods 110 cases of patients with ventilator associated pneumonia was selected in a hospital from October 2014 to October 2016 as pathogenic groups .At the same time ,patients treated with ventilator adjuvant therapy for the same period without infection as non-pathogenic group (120 cases) ,selecting the same period of physical examination no abnormal patients as the healthy group (95 cases) ,detection and analysis of pathogenic characteristics in patients with ventilator associated pneumonia , detection of the serum levels of PCT ,CRP and lung function .Analyzed the correlation with the changes of ser-um PCT ,CRP and pulmonary function .Results 210 strains of pathogens were isolated from the secretions of 110 patients with pathogenic groups ,among them ,90 strains of Gram-positive bacteria accounted for 42 .85%, Gram-type negative bacteria accounted for 119 cases accounted for 56 .67% and a strain of fungus .The levels of PCT and CRP in the non-pathogenic group and pathogenic group were significantly higher than those in the healthy group ,the difference was statistically significant (P＜0 .05) .Serum PCT and CRP levels in the patho-genic group were significantly higher than those in the non-pathogenic group and the healthy group ,the differ- ence was statistically significant (P＜0 .05) .Logistic multiple regression analysis was used to analyze the inde-pendent risk factors of ventilator-associated pneumonia in patients .The duration of invasive ventilation ,gram-negative oropharyngeal bacteria count ,age and past history of COPD were independent risk factors ,with sig-nificant difference (P＜0 .05) .Patients in the pathogenic group had a forced expiratory volume (FEV1) ,forced vital capacity (FVC) ,forced expiratory force occupancy force ratio (FEV1/FVC) ,the first second percentage of forced vital capacity occupies vital capacity (FEV1%)were significantly higher than those in the non-patho-genic group and the healthy group ,the difference was statistically significant (P＜0 .05) .FEV1 ,FVC ,FEV1/FVC and FEV1% in the non-pathogenic group and pathogenic group were significantly higher than those in the healthy group ,the difference was statistically significant (P＜0 .05) .Conclusion Gram-negative bacteria predominate in ventilator-associated pneumonia .
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Objective Establish detection method to measure Vibrio vulnificus rapidly and accurately.MethodsUsing flow cytometry(FCM)and a 5'-FITC fluorescent labeled aptamer with high binding affinity to detect Vibrio vulnificus rapidly.Measure a series of concentrations of Vibrio vulnificus to identify the Limit of Blank, Lower Limit of Detection, Linearity Range, etc.ResultsCombined application of FCM and the aptamer can detect Vibrio vulnificus rapidly with the duration less than 1 hour and lower limit of detection as low as 29 CFU/mL.Conclusion The aptamer targeting Vibrio vulnificus is an excellent detective element, while FCM can realize accurate quantitative detection.The detection method has great application potential.
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Rapid detection of infection pathogens is of great importance to the prevention and control of infectious diseases.Compared with traditional approaches,point-of-care testing (POCT) technologies promise great advantages in simple, rapid and portable detection of pathogens.In this review, the technologies, categories, developments and applications of POCT in detection of infectious pathogens are elaborated.Furthermore, the future developments of POCT detection of infectious pathogen are also discussed.This review focuses on loop-mediated isothermal amplification ( LAMP) technology, microfluidic chip and biosensor technology in the POCT detection of infectious pathogens while elaborating on the application of these new technologies associated with POCT detection.
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Objective To improve microbial specimen detection rate before therapeutic antimicrobial use.Methods A system of selective targeted management by clinical department was established,before management was as control group (July-September 2013),after management was as intervention group(October-December 2013),microbial specimen detec-tion in patients before antimicrobial use was compared between before and after management.Results Of all hospitalized pa-tients,11 254 received therapeutic antimicrobial agents,3 426 were sent specimens for microbial detection,the specimen detection rate was 30.44%;specimen detection rate in control and intervention group was 28.80% and 31.89% respective-ly ,the difference was significant(χ2 =12.71,P <0.05).3 716 patients(46.61%)received restrained antimicrobial therapy, and 1 418 (79.20%)received special antimicrobial therapy,compared with control group,the difference were both signifi-cant(χ2 =32.86,19.31,respectively,both P <0.05).Conclusion Applying selective targeted management can improve microbial specimen detection rate before therapeutic use of antimicrobial agents.
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Objective To analyze the epidemiological characteristics of pathogens of children with hand , foot and mouth disease (HFMD) in Nantong area, in order to provide the basis for the treatment and prevention of HFMD. Methods Multiplex RT-PCR (fluorescence PCR method) was used to detect EV71, CoxA16 and non-infectious cases of EV71, CoxA16 of children with HFMD in Nantong. VP1 gene of strains isolated from 12 cases of EV71 was amplified and underwent phylogenetic tree analysis. 208 cases clinical data of children with HFMD were analyzed and summarized. Results In 208 cases of children with HFMD swab samples, EV71 of 64 cases were positive (30.8%); CoxA16 of 28 cases were positive (13.5%); 40 cases of other enterovirus were positive (19.2%). Phylogenetic tree analysis showed that EV71 epidemic strains are C4 subtypes in Nantong region. The clinical manifestations of enterovirus infections in EV71 , CoxA16 and other enterovirus are caused by rash, fever and other symptoms, the difference of which was not statistically significant (P > 0.05). In laboratory examination, EV71 infection made white blood cell count higher than CoxA16 and other enterovirus, but the difference was not statistically significant (P>0.05). Conclusions EV71, CoxA16 and other enterovirus intestinal virus are main pathogens of HFMD in Nantong. EV71 C4 subtype is the main popular subtype of EV71 in Nantong. The elevation of white blood cell count of HFMD is the main clinical manifestations and characteristics , but can not serve as the identification of children infected with EV71, CoxA16 and other enterovirus.
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Objective To explore a rapid bacterial identifying method based on the 16S rRNA gene sequence analysis technology to provide the scientific basis for the diagnosis and treatment of unknown pathogenic bacteria.Methods The pure colonies were iso-lated and cultured directly from a clinical patient′s sputum sample.The colony as a template for PCR amplification with universal primers to amplify 16S rRNA gene fragments of unknown bacteria.The product of PCR was sequenced directly,then the sequence result was compared by using the BLAST of NCBI and the pathogen was identified based on the sequence homology.Results 1 strain of unknown pathogen was identified as ochrobactrum by this test and confirmed by ABI bacterial rapid identification sys-tem.Conclusion This study simplifies the isolation and identification procedures of unknown pathogen from the clinical samples and establishes a simple method for the rapid identification of pathogens by using 16S rRNA gene amplification.
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Objective To develop a sensitive,specific, simple and rapid quantitative real-time PCR (Q-PCR) assay for detection of Staphylococcus aureus with SmartCycler.Methods According to the nuc gene sequences specific to S.aureus, a pair of primers and one TaqMan probe were designed. An internal amplification control (IAC) which is a chimeric double-stranded DNA constructed from a fragment of the Listeria monocytogenes hly gene flanked by the nuc-specific target sequences was added to the reaction system. This IAC was detected using a second TaqMan probe labeled with a different fluorophore. The performance of the nuc-IAC Q-PCR was evaluated using artificially contaminated drinking water and commercial UTH whole milk samples spiked with ATCC 6538.Results The nuc-IAC assay could be used reliably for detection with a sensitivity of 5 copies of linear plasmid DNA per reaction, 10 fg of genomic DNA in 62.5% of the reactions or 50 cfu/ml S.aureus cells with 50% probability. The quantification was linear (r~2≥0.998) over a 6-log dynamic range, with a PCR efficiency over 0.967. The 5×10~2 CFU per 25 ml mimic sample of drinking water or milk could be detected by this assay consistently and quantifiably.Conclusion The nuc-IAC Q-PCR assay for S.aureus is developed. It could not only be applied for the quantitative detection of S.aureus, but also prevent the false negatives and underestimations of contamination loads due to PCR failure.
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Objective To investigate the distribution, composition and situation of natural infection pathogen of tick species in the main ports of Inner Mongolia. Methods All ticks were collected manually with white cloth, from the grassland and searching for the hosts followed by detection of pathogens, with PCR. Results 1313 ticks identified, belonged to 1 family,4 geniuses and 7 species in the three surveyed areas, with Dermacentor nuttallia distributed in the Ceke, Mandula and Manzhouli bordering ports. 69.08% of the total species were discovered at Port Ceke, with Rhipicephalus pumilio as the predominant one, which accounted for 74.86%. 5 kinds of tick-borne disease pathogens were detected from ticks in these three bordering ports while only Coxiella burnetii was found at the Port Ceke. In these three ports, the average infection rates of Lyme disease borrelia , Human babesia microti, Spotted fever group Rickettsia, Caxiella burnetii, Ehrlichiosis were 15.08%, 3.35%, 1.98%, 1.07%, 0.99% respectively.The positive rate of tick infected with Borrelia burgdorferi were 13.56%, 22.88%, 5.00% in the 3 bordering ports, respectively with significant differences. The positive rates of Babesia microti and Spotted fever group Rickettsia infections were also significantly different among these areas. Conclusion The natural infection rates of the above mentioned five kinds of tick-borne pathogens were different in the Ports Ceke,Mandula and Manzhouli.