Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction （PCR） and capillary electrophoresis （MPCE） assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catorrholis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebactefium diphthefiae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 252 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE with high specificity and sensitivity. This assay survey of respiratory pathogens. assay is a rapid, reliable, and high-throughput method has great potential in the molecular epidemiological.

Porcine Respiratory Pathogens in Swine Farms Environment in Mexico

Respiratory pathogens are the main health problem in the swine industry worldwide. These pathogens are transmitted by direct contact between animals or by aerosols and however are not well known yet, if the environment works as its reservoir, inoculum and/or dispersion medium. The objective of this study was to determine the presence of respiratory pathogens in environmental samples from swine farms in Aguascalientes, Mexico, through of PCR and RT-PCR techniques. The bacteria Actinobacillus pleuropneumoniae and Pasteurella multocida were found viable in samples from water, food, soil and air. Streptococcus suis was found in a viable state in water samples. Haemophilus parasuis, Porcine Reproductive and Respiratory Syndrome virus and Swine Influenza virus (H1N1 and H3N2) were detected in drinking water samples. Mycoplasma hyopneumoniae and Porcine Circovirus type 2 (PCV2) were not detected in environmental samples. These results suggest that the environment of the farms acts as a reservoir, inoculum and/or vehicle of dispersion for these pathogens except for M. hyopneumoniae and PCV2.

Traditional Chinese Medicine Nursing Protocols for Fever Caused by Exogenous Pathogens (Upper Respiratory Infection)

Fever caused by exogenous pathogens,also called upper respiratory infection( URI),refers to a classification of exogenous diseases induced by invasion of six pathogenic factors into the body leading to disharmony betw een nutrient Qi and defensive Qi and imbalance of Yin and Yang. As an extremely common symptom or sign,it is frequently seen in various processes of acute and chronic diseases. In recent years,traditional Chinese medicine( TCM) has achieved a certain efficacy in the treatment of fever caused by exogenous pathogens,such as modified formulae,Chinese patent drugs,self-made formulae,acupuncture,moxibustion and so on.This article mainly explored the key points of common syndromes,TCM nursing methods and health guidance of fever caused by exogenous pathogens,in order to further develop the advantages of TCM,improve its efficacy and standardize its nursing behavior.

Location of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Tissues of Natural Cases

[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome （HP-PRRS）. [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.

Evaluation of the Pathogenicity of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant in Piglets

Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China＇s pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses （3×103-3×105 TCID50）.The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever （41.0-41.9oC） and high morbidity and mortality （60-100%）,the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection （DPI）.The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-g and IL-10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.

Tracking Survey of Immune Effects of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome (HP-PRRS) in Changping District, Beijing City

The epidemiology of highly pathogenic porcine reproductive and respiratory syndrome( HP-PRRS) in Changping District,Beijing City was investigated from 2010 to 2015. Through statistics of vaccine,immune procedure and antibody levels,the epidemic prevention effect of HP-PRRS in Changping District was scientifically assessed.

A direct real-time polymerase chain reaction assay for rapid high-throughput detection of highly pathogenic North American porcine reproductive and respiratory syndrome virus in China without RNA purification

Background： Porcine reproductive and respiratory syndrome virus （PRRSV）, and particularly its highly pathogenic genotype （HP-PRRSV）, have caused massive economic losses to the global swine industry. Results： To rapidly identify HP-PRRSV, we developed a direct reaL-time reverse transcription polymerase chain reaction method （dRT-PCR） that could detect the virus from serum specimen without the need of RNA purification Our dRT-PCR assay can be completed in 1.5 h from when a sample is received to obtaining a result. Additionally, the sensitivity of dRT-PCR matched that of conventional reverse transcription PCR （cRT-PCR） that used purified RNA The lowest detection limit of HP-PRRSV was 6.3 TCIDs0 using dRT-PCR. We applied dRT-PCR assay to 144 field samples and the results showed strong consistency with those obtained by cRT-PCR. Moreover, the dRT-PCR method was able to tolerate 5-20% （v/v） serum. Conclusions： Our dRT-PCR assay allows for easier, faster, more cost-effective and higher throughput detection of HP-PRRSV compared with cRT-PCR methods. To the best of our knowledge, this is the first report to describe a real-time RT-PCR assay capable of detecting PRRSV in crude serum samples without the requirement for purifying RNA. We believe our approach has a great potential for application to other RNA viruses.

Pathogen Distribution and Laboratory Diagnosis of Acute Respiratory Infectious Diseases

Objective: To investigate the etiology distribution and laboratory diagnosis of acute respiratory infectious diseases. Methods: By searching, collecting and summarizing the etiology distribution and laboratory diagnosis of respiratory infectious diseases, the latest research progress of acute respiratory infectious diseases was studied in order to provide reference for clinical prevention and treatment. Results: In the relevant literature collected, it is considered that acute respiratory infectious diseases refer to infectious diseases caused by pathogens invading human body from the nasal cavity, throat, trachea and bronchus. The main acute respiratory infectious diseases are SARS (acute severe respiratory syndrome), MERS (Middle East respiratory syndrome), 2019-ncov infection (new coronavirus pneumonia), pulmonary plague, influenza, measles, diphtheria, pertussis, rubella, meningitis, mumps, tuberculosis, etc. Different types of infection have different diagnostic methods, and different treatments are given according to different diseases. Conclusion: The etiology distribution and laboratory diagnosis of acute respiratory tract infectious diseases are understood through reference, which can provide better reference for clinical practice. Acute respiratory infectious disease is the most common clinical disease, which seriously endangers people’s health.

Molecular Characterization of a Highly Pathogenetic Porcine Reproductive and Respiratory Syndrome Virus Variant in Hubei, China

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.

Prevention and Treatment Strategy of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome(HP-PRRS)

At the present stage, the social economy in China has developed by leaps and bounds. China's pig industry has been well developed un-der the background of this era. However, the number of pig diseases increases due to the influence of many factors, and highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS) is one of the very common diseases, causing growing mortality rate of pigs. The disease increases the economic loss of pig industry to a large extent, and brings difficulties in prevention and control of pig diseases. The staff must analyze and sum-marize the basic characteristics of pig diseases, and combine some practical problems in prevention and control of pig disease to find effective mea-sures. The scope and scale of pig production in different parts of China have been continuously expanded under the background of further develop-ment of pig industry in the new era. But for pig production, pig disease has been a problem that delays its further development. Related personnel must analyze and summarize the status quo of HP-PRRS, to find effective prevention measures and strategy.

Analysis of Common Respiratory Infected Pathogens in 3100 Children after the Coronavirus Disease 2019 Pandemic

Objective To investigate the epidemiological features in children after the coronavirus disease 2019(COVID-19)pandemic.Methods This study collected throat swabs and serum samples from hospitalized pediatric patients of Renmin Hospital of Wuhan University,Wuhan,Hubei province,China before and after the COVID-19 pandemic.Respiratory infected pathogens[adenovirus(ADV),influenza virus A/B(Flu A/B),parainfluenza virus 1/2/3(PIV1/2/3),respiratory syncytial virus(RSV),Mycoplasma pneumoniae(MP),and Chlamydia pneumoniae(CP)]were detected.The pathogens,age,and gender were used to analyze the epidemiological features in children after the COVID-19 pandemic.Results The pathogen detection rate was significantly higher in females than in males(P<0.05),and the infection of PIV1 and MP was mainly manifested.After the COVID-19 pandemic,PIV1,PIV3,RSV,and MP had statistically different detection rates among the age groups(P<0.05),and was mainly detected in patients aged 0–6 years,0–3 years,0–3 years,and 1–6 years,respectively.When comparing before the COVID-19 pandemic,the total detection rate of common respiratory pathogens was lower(P<0.05).Except for the increase in the detection rate of PIV1 and CP,the infection rate of other pathogens had almost decreased.Conclusion The prevention and control measures for the COVID-19 pandemic effectively changed the epidemiological features of common respiratory tract infectious diseases in pediatric children.

优化监测、防控与免疫策略应对呼吸道传染病季节性高发流行

新型冠状病毒感染大流行之后,呼吸道病原体流行强度和特征改变,耐药问题日益凸显,呼吸道病原体防控愈加紧迫。本期重点号以"呼吸道病原防控"为主题,组织多学科专家针对多种呼吸道病原体交替流行的现状,总结各种病原体流行特征、疫苗研发及耐药研究现状,提出应对策略,强调创新医防协同医防融合机制及措施,开展多病原监测与多病共防,提升我国呼吸道传染病防控能力,优化疫苗策略和免疫服务,提高诊疗和管理规范性,加大脆弱人群的保护力度,降低疾病负担。

2023-2024年南京及周边地区儿童常见6种呼吸道病原体感染的流行病学研究

目的 研究南京市及其周边地区2023年5月至2024年4月期间儿童呼吸道病原体感染性疾病的流行病学及其特征,确定病原体感染的种类,为呼吸道感染性疾病的预防和治疗提供依据。方法 回顾性分析南京市儿童医院2023年5月至2024年4月的88437例患儿标本,采用荧光PCR的方法检测6种病原体,分别为腺病毒(ADV),甲型流感病毒(FluA),乙型流感病毒(FluB),人鼻病毒(HRV),肺炎支原体(MP)和呼吸道合胞病毒(RSV),并对结果进行统计分析。结果 88437个样本阳性检出率为68.98%,六种病原体检出率由高到低分别MP25.77%,ADV15.24%,HRV12.18%,RSV11.51%,FluA8.25%,FluB7.24%。各病原体不同年龄段之间的检出率差异有统计学意义,其中7~18岁儿童检出率最高,时间因素对此也有影响,秋冬季的阳性率较高。其中MP,Flu A与Flu B的检出率存在交替高峰,此消彼长的状态。不同病原体中混合感染的阳性率也较高,其中最常见为HRV与MP混合感染。结论 儿童呼吸道病原体感染以MP感染最为常见,其中MP,Flu A与Flu B存在交替高峰的特点,混合感染以HRV与MP最为常见,不同年龄、时间对病原体感染均有影响。病原体感染多发于7~18岁儿童,应加强对7~18岁儿童的呼吸道感染知识的科普教育。秋冬季为呼吸道病原感染的高发季节,需加强秋冬季节的防护与监测。

新发突发呼吸道传染性病原微生物分级检测专家共识(2024)

近年来,呼吸道传染性疾病不断暴发流行,给全球公共卫生和医疗卫生体系带来了巨大挑战。如何及时采取分级筛查和检测策略,系统覆盖引起呼吸道急性感染的临床常见传染性病原体、慢性感染或少见呼吸道传染性病原体,着眼可能的新发突发呼吸道传染性病原体,快速明确致病微生物,成为临床诊断亟需解决的问题。为此,中国医院协会临床微生物实验室专业委员会组织多学科专家,总结目前国内实验室检测在临床实践中所积累的应用经验,结合当前国际最新研究进展,共同编写了《新发突发呼吸道传染性病原微生物分级检测专家共识(2024)》。本共识通过分析重大新发突发呼吸道病原体传染性和感染特点,兼顾患者人群特征,制定了包含传统微生物形态学、培养、免疫学、分子生物学以及宏基因组测序等方法的三级检测策略,明确了在不同阶段针对的目标病原体及推荐检测方法,为提高新发突发呼吸道传染病的诊断能力提供了指导建议和参考依据。

不同年龄段呼吸道感染住院患儿的病原体核酸检测结果及混合感染情况分析

目的探讨不同年龄段呼吸道感染住院患儿的病原体核酸检测结果及混合感染状况分析。方法回顾分析2021年1月至2023年12月于郑州市妇幼保健院住院治疗的130例呼吸道感染患儿临床资料,所有患儿均展开核酸呼吸道合胞病毒(RSV)、腺病毒(ADV)、流感病毒A和B型(IFA、IFB)、人鼻病毒(HRV)、肺炎支原体(MP)检测。分析不同年龄段、性别各病原体阳性检出率,另探讨患儿呼吸道患儿混合感染状况。结果经统计分析,130例呼吸道感染患儿中共检出79例病原体核酸阳性患者,占60.77%,呼吸道病原体检出率从高至低依次为MP、IFB、RSV、IFA、ADV、HRV。通过分析不同年龄段患儿病原体核酸阳性率,结果显示7~10岁患儿阳性率最高,其次为4~6岁,其中<1岁患儿以RSV感染为主,1~3岁以IFA感染为主,4~6岁患儿以MP感染为主,7~10岁患儿以IFB感染为主。女性患儿病原体阳性率为62.90%,高于男性患儿58.82%,但两者比较差异无统计学意义(P>0.05)。单一病原体核酸阳性患儿共63例(79.75%),双重病原体感染患儿15例(18.99%);仅1例三重病原体感染,为IFB+MP+RSV。结论呼吸道感染住院患儿感染病原体主要为MP、IFB、RSV三种,7~10岁患儿阳性率最高,其次为4~6岁患儿,女性较男性患儿更易感染,混合感染以MP+IFB多见。

探究2023年颍淮地区内非重症呼吸道感染者病原体流行特征及现况调查

目的 回顾性分析颍淮地区内非重症呼吸道病原体感染情况的流行病学特征。方法 纳入2023年1月—2023年12月界首市中医院、安徽中医药大学附属太和中医院1238例具有典型呼吸道感染症状的患者,通过20余种病原学检查,回顾性探究颍淮地区内呼吸道感染患者流行病学特征及现况调查。结果 呼吸道病原体总体检出阳性率为59.94%(742/1238),肺炎支原体检出阳性率为49.35%(611/1238),占阳性病例达82.35%(611/742);细菌阳性率为17.69%(219/1238),占29.51%(219/742);病毒阳性率为12.52%(155/1238),占比20.89%(155/742);在所有病原体种类里,排序前5类为肺炎支原体611例、甲型流感病毒109例、乙型流感病毒84例、肺炎克雷伯杆菌68例、铜绿假单胞菌68例;单一病原体感染占阳性病例的71.83%(533/742),混和感染有28.17%(209/742)。在11、3、10、12月检出率较高(P<0.05),实年组和老年组的检出率较低(P<0.05)。无序多分类Logistic回归模型分析结果提示年龄、性别、发病时间3个因素对感染数量均有不同程度的影响。结论 2023年1月-2023年12月颍淮地区内非重症呼吸道传染病感染病原体的阳性检出率较高,病原体感染以肺炎支原体为主,细菌及病毒感染情况减少,有部分患者存在混和感染情况。冬春季节病原体的阳性检出率较高,年长者检出率较低,且时间、年龄、性别会影响病原体混和感染率。

急性上呼吸道感染患者病原体种类与临床特征分析

目的分析急性上呼吸道感染患者病原体种类与临床特征。方法选取2023年12月1日至2024年5月15日在南京大学医学院附属金陵医院收治的急性上呼吸道感染患者275例作为研究对象。采用实时荧光定量聚合酶链反应技术检测咽拭子标本的病原体种类,以及收集病历系统中患者临床资料和外周血检验指标。结果甲型流感病毒阳性检出率9.45%,乙型流感病毒阳性检出率17.82%,呼吸道合胞病毒阳性检出率9.45%,腺病毒阳性检出率9.21%,新型冠状病毒阳性检出率18.37%,肺炎支原体阳性检出率10.04%,肺炎衣原体阳性检出率1.67%。与其他年龄段患者比较,<18岁患者甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体阳性检出率较高,差异有统计学意义(P<0.05)。乙型流感病毒感染患者单核细胞比例高于其他病原体感染患者,差异有统计学意义(P<0.05)。结论急性上呼吸道感染患者病原体种类的确定,有助于为患者制订适当的治疗方案,为患者的临床诊疗提供依据。

黑龙江齐齐哈尔地区牛呼吸道疾病综合征重要病原体流行病学调查

[目的]了解黑龙江省齐齐哈尔地区牛呼吸道疾病综合征重要病原体流行与病原菌的耐药情况,为齐齐哈尔地区牛呼吸道疾病综合征的防治提供依据。[方法]采集齐齐哈尔不同地区规模化养殖场及散养户发病牛的鼻拭子样本594份,应用PCR检测样本中携带的牛呼吸道疾病综合征病原体,并进行细菌分离培养及药敏试验。[结果]594份鼻拭子样品中共有209份样品携带牛呼吸道疾病综合征病原体,其中牛支原体检出率为33.01%、多杀性巴氏杆菌检出率为15.79%、溶血性曼氏杆菌检出率为16.75%、牛传染性鼻气管炎病毒检出率为15.79%、牛病毒性腹泻病毒检出率为20.57%。通过细菌分离培养获得多杀性巴氏杆菌27株、溶血性曼氏杆菌26株。通过药敏试验发现多杀性巴氏杆菌和溶血性曼氏杆菌三重耐药菌株占比分别为51.85%和38.46%,四重耐药菌株占比分别为22.22%和23.08%,五重耐药菌株占比分别为22.22%和15.38%,六重耐药菌株占比分别为3.70%和23.08%。[结论]黑龙江省齐齐哈尔地区引起牛呼吸道疾病综合征的重要病原体检出率较高,多杀性巴氏杆菌和溶血性曼氏杆菌菌株多重耐药情况较为严重。

Clinical Diagnosis and Treatment Characteristics of Acute Respiratory Infections in Children and New Developments in Laboratory Testing

Acute respiratory tract infection (ARTI) in children is the most common infectious disease in childhood, and its pathogens include viruses, bacteria and fungi, mycoplasma, chlamydia and rickettsia. In recent years, with the continuous development of pathogen detection methods, the diagnosis and treatment of acute respiratory infections has received more and more clinical attention. The clinical diagnosis and treatment characteristics of acute respiratory infections in children and the research of clinical laboratory detection methods have also been continuously developed. The author collected references to review the clinical features and new developments in laboratory testing of acute respiratory tract infection in children.

Monitoring of Respiratory Infections in the Intensive Area of Respiratory System Disease

Respiratory infections are the most frequent nosocomial infections after those urinaries and surgicals. We analysed respiratory infection incidences in patients treated with different kinds of respiratory assistance (non-invasive ventilation and invasive mechanical ventilation trough tracheostomy), studying 640 patients recovering in the intensive area of respiratory disease from 2010 to 2013. We had 113 cases of respiratory infections: 42.5% in patients of non-invasive ventilation group and 57.5% of patients treated with invasive ventilation with a statistically significant difference between the two groups. The patients treated with non-invasive ventilation showed a lower incidence of nosocomial respiratory infections. Prevention guidelines are important to reduce nosocomial infections frequency.