Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

The future of new oral antibiotics including the quinolones.

It is estimated that more than 110 million dollars'' worth of oral antibiotics will have been sold in Canada in 1987. In the next few years several new oral antimicrobial agents will reach the market, including beta-lactamase inhibitors, cephalosporins, monobactams, erythromycins and quinolones. Most of these new agents have a broader spectrum of antibacterial activity than the presently available oral antibiotics. A few have a longer half-life and can be administered once a day. The new oral drugs, especially the quinolones and possibly beta-lactams, will now be used to treat infections that in the past could be treated only parenterally. Exacerbations of pulmonary infections due to Pseudomonas aeruginosa in cystic fibrosis can now be successfully treated at home with the new quinolones. Osteomyelitis, arthritis, pneumonia and pyelonephritis will most likely be treated at home in the future. In severe infections patients will be admitted to hospital for short courses of parenteral therapy, followed by oral treatment. If used appropriately the new oral agents may lead to new approaches to the treatment of infectious diseases.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

提高临床医学生微生物检验 见习教学质量的探索与实践

临床微生物室见习是临床医学专业学生了解微生物检验的重要窗口,提高其见习教学质量,使其认识到微生物检验在感染性疾病诊治中的作用。除了系统介绍临床微生物检验的工作内容及流程外,我们更注重临床医生与微生物检验的密切联系,强调临床医生对微生物检验前质量控制的重要性。案例教学法联合问题导向教学法,将临床感染病例与微生物检验紧密结合。举例讲解临床医生该如何正确解读微生物检验报告结果,从而合理选择抗菌药物进行有效的抗感染治疗。最后提醒临床医学生注意生物安全及医院感染的发生。通过我们的见习教学,临床医学生表示获益颇丰,意识到微生物检验对感染性疾病诊治的重要作用。     

Enzyme immunoassays for the detection of microbial antigens and prospects for improved assays

The rapid diagnosis of viral infections is an important tool in the management of patients with infectious diseases. Solid-phase enzyme immunoassays have proved to be useful tools for the direct detection of the antigens of some viruses directly in clinical specimens. Such assays have been particularly useful in the diagnosis of viral infections in the gastrointestinal and respiratory tracts. However, standard solid-phase enzyme immunoassays often do not display sufficient sensitivity for the diagnosis of all cases of viral infections. Techniques which might be utilized to increase the sensitivity of solid-phase immunoassays include the use of monoclonal antibodies to maximize the efficiency of the antigen-antibody interactions and the use of high-turnover enzymes to increase the amount of signal generated by the ensuing enzyme-substrate reactions. In addition, techniques making use of nucleic acid hybridization have a great deal of potential for the accurate detection of viral nucleic acids in human body fluids. The successful application of these techniques to the diagnosis of viral infections could lead to a marked improvement in the care of patients with suspected infectious diseases as well as to a decrease in the transmission of viral infections to high-risk individuals.     

Apoptosis in the pathogenesis of infectious diseases of the eastern oyster Crassostrea virginica

Apoptosis, or programmed cell death, has been reported as being pivotal in infectious diseases of different organisms. The effects of apoptosis on the progression and transmission of the protistan parasites Perkinsus marinus and Haplosporidium nelsoni in the eastern oyster Crassostrea virginica were studied. Oysters were diagnosed for their respective infections by standard methods, and apoptosis was detected using in situ hybridization to detect DNA fragments by end labeling on paraffin sections. A digoxigenin nucleotide probe was used to label the 200 bp fragment produced by apoptosis and detected immunohistochemically using an antidigoxigenin peroxidase conjugate. The probe/DNA fragment complex was stained with a peroxidase substrate and tissues were counterstained with methyl green. Uninfected oysters had large numbers of apoptotic hemocytes present in the connective tissue underlying the stomach, gill, and mantle epithelia, whereas oysters infected with P. marinus had a reduced number of apoptotic hemocytes. The parasite may prevent hemocyte apoptosis in order to yield a greater number of hemocytes in which to house itself. Large numbers of P. marinus cells in some infected oysters were eliminated via apoptosis in the stomach epithelia, disabling the spread of infectious particles through seawater. The oysters infected with H. nelsoni also had reduced numbers of apoptotic hemocytes, while part of the vesicular connective tissue cells were apoptotic. H. nelsoni plasmodia were eliminated via apoptosis in some oysters. Apoptosis may enhance progression and prevent transmission of infectious oyster diseases.     

感染性和非感染性发热的临床特征与鉴别

目的分析感染性和非感染性发热的鉴别诊断。方法收集1997年1月。2003年3月收住的以发热为主要临床表现的患者706例,进行回顾性分析。结果感染性疾病550例（81．12％）,其中局灶感染203例（29．94％）,多系统性感染347（51．18％）。非感染性疾病107例（15．78％）。结论感染性和非感染性发热的鉴别诊断在于鉴别诊断思路的完整性,综合衡量是诊断的方法。     

感染组学 (infectomics) ———对感染性疾病的总体性和综合性研究

在感染性疾病的范畴内,目前急需一个能有效地、精确地和综合性地研究微生物感染的结构性和功能性基因组学和蛋白质组学 ( 感染组学 ) 的全面方法. 新的方法 ( 如 DNA 和蛋白质微阵列 ) 和传统方法 ( 如分子克隆、 PCR 、基因敲除,加进 (knockin) 和反义术等 ) 的结合将有助于克服今天的困难. 在感染时,微生物及其宿主的全部表型改变 ( 感染组 ) 均由微生物病原体及其宿主的基因组所编码,并在特异的微生物 - 宿主相互作用时的某些环境条件下表达. 微生物及其宿主的全部药物反应 ( 药理组 ) 可用基因组或蛋白质组的方法检出. 分析基因型和表型或表达形式的全基因组方法将最终导致对微生物的发病机理、感染性疾病的快速诊断和控制感染的新策略的全面研究. 感染性疾病中最基本的问题是,如何全面地和综合性地应用感染组学,来了解微生物病原体及其宿主的相互作用.     

口腔微生物多样性研究进展

口腔微生物多样性的改变是口腔常见感染性疾病的发病机制之一,随着分子生物学技术的普遍应用,研究者能够不通过纯培养技术即可对人类口腔不同生态系的微生物组成及变化进行研究。本文综述了当前口腔微生物多样性研究的常用方法及各自的特点,从而为口腔微生物生态方面的进一步深入研究提供参考。     

Cutaneous infection by Phaeoacremonium parasiticum

BackgroundPhaeoacremonium parasiticum is considered a rare infectious agent that is part of a heterogeneous group of fungi causing phaeohyphomycosis. This organism is capable of producing subcutaneous infections, eumycetomas, osteomyelitis, arthritis, myositis and also disseminated diseases, such as fungemia and endocarditis.Case reportWe describe a case of cutaneous infection by P. parasiticum in a kidney transplant patient. The identification of this microorganism was performed by microbiological and histopathological studies and confirmed with the sequence of the gene encoding β-tubulin and a real time panfungal PCR targeting 18S ribosomal RNA gene. The microorganism was correctly identified by phenotypic and molecular methods. The patient was treated with oral antifungal therapy and a debulking surgery and evolved without any complication.ConclusionsThe diagnosis of this infection is difficult and usually affects kidney transplant patients, but the reasons of this association are still unknown.     

侵袭性真菌病的诊断：现状与展望

近二十年来,医学科学很多领域都取得重大进步。但全球范围内,侵袭性真菌病的发病率及死亡率却仍明显上升,严重威胁人类健康。侵袭性真菌病发病隐匿、临床表现不典型、治疗手段有限、病死率与致残率高,早期、特异的诊断对于改善预后意义重大。目前,以培养、病理为代表的形态学诊断方法虽有局限,但仍是侵袭性真菌病诊断的金标准;以G试验、GM试验、高分辨率CT为代表的新兴血清学及影像学诊断方法值得在临床大力推广;而以PCR技术为基础的核酸诊断技术方法前景光明,但其临床应用之路却仍任重而道远。联合使用并不断改良现有培养、病理等形态学诊断方法、血清学方法及先进影像学技术是提高侵袭性真菌病诊断水平的现实最佳途径。     

Intérêt de l’imagerie nucléaire pour l’infectiologue

Nuclear imaging could be helpful for infections diseases specialists in search for bone or joint and endovascular septic foci (with or without foreign body) but also for various metastatic infectious locations and for exploring fever of unknown origin (FUO), especially in HIV-infected patients. In this paper, the interest of “conventional” scintigraphic methods (labeled leukocyte and gallium scintigraphy) and positron emission tomography coupled with fluorodeoxyglucose (FDG–PET) for the diagnosis of endovascular infections (especially those associated with foreign body) and some opportunistic diseases in HIV-infected patients (especially those presenting central nervous system involvement and FUO) will be discussed. Then other potential areas of isotopic investigations will be briefly listed; some of them, including bone and joint infections and FUO in the general population, will be presented in details in other papers in this issue.     

宏基因组测序在中枢神经系统感染性疾病诊断中的应用及研究进展

中枢神经系统(central nervous system,CNS)感染是指由病毒、细菌或真菌等侵染中枢神经系统引起的急性或慢性炎症性(或非炎症性)疾病,致死率高,易引发严重后遗症。由于检测通量及灵敏度的限制,一半以上的中枢神经系统感染患者无法通过常规检测方法确定病原体。宏基因组测序是一种新兴的病原检测技术,能够极大地提升病原检出率。当前部分临床医生及相关从业人员对宏基因组测序的认识存在不足,限制了其在临床诊疗中的快速推广和应用。本文系统介绍了宏基因组测序整体流程,综述了该技术在中枢神经系统感染性疾病诊疗中的发展历程和最新研究进展,希望为中枢神经系统感染性疾病的诊断和治疗提供参考。     

Evaluation of Anti-A/Udorn/307/1972 Antibody Specificity to Influenza A/H3N2 Viruses Using an Evanescent-Field Coupled Waveguide-Mode Sensor

Discrimination of closely related strains is a key issue, particularly for infectious diseases whose incidence fluctuates according to variations in the season and evolutionary changes. Among infectious diseases, influenza viral infections are a worldwide cause of pandemic disease and mortality. With the emergence of different influenza strains, it is vital to develop a method using antibodies that can differentiate between viral types and subtypes. Ideally, such a system would also be user friendly. In this study, a polyclonal antibody generated against A/Udorn/307/1972 (H3N2) was used as a probe to distinguish between influenza H3N2 viruses based on the interaction between the antibody and hemagglutinin, demonstrating its applicability for viral discrimination. Clear discrimination was demonstrated using an evanescent-field-coupled waveguide-mode sensor, which has appealing characteristics over other methods in the viewpoint of improving the sensitivity, measurement time, portability and usability. Further supporting evidence was obtained using enzyme-linked immunosorbent assays, hemagglutination-inhibition assays, and infectivity neutralization assays. The results obtained indicate that the polyclonal antibody used here is a potential probe for distinguishing influenza viruses and, with the aid of a handheld sensor it could be used for influenza surveillance.     

Applications of sequencing technology in clinical microbial infection

Infectious diseases are a type of disease caused by pathogenic microorganisms. Although the discovery of antibiotics changed the treatment of infectious diseases and reduced the mortality of bacterial infections, resistant bacterial strains have emerged. Anti‐infective therapy based on aetiological evidence is the gold standard for clinical treatment, but the time lag and low positive culture rate of traditional methods of pathogen diagnosis leads to relative difficulty in obtaining the evidence of pathogens. Compared with traditional methods of pathogenic diagnosis, next‐generation and third‐generation sequencing technologies have many advantages in the detection of pathogenic microorganisms. In this review, we mainly introduce recent progress in research on pathogenic diagnostic technology and the applications of sequencing technology in the diagnosis of pathogenic microorganisms. This review provides new insights into the application of sequencing technology in the clinical diagnosis of microorganisms.     

Infectomics: genomics and proteomics of microbial infections

The completion of genomic sequences is the greatest triumph of molecular reductionism since the discovery of the DNA double helix in 1953. However, the utility of reductionism is becoming limited and holistic approaches, including theories and techniques, are desperately needed in the postgenomic era. In the field of infectious diseases there is an urgent need for global approaches that can efficiently, precisely and integratively study structural and functional genomics and proteomics of microbial infections (infectomics). The combination of new (e.g. DNA and protein microarrays) and traditional approaches (e.g. cloning, PCR, gene knockout and knockin, and antisense) will help overcome the challenges we are facing today. We assume that the global phenotypic changes (infectomes) in microbes and their host during infections are encoded by the genomes of microbial pathogens and their hosts, expressed in certain environmental conditions devoted to specific microbe-host interactions. Global drug responses (pharmacomes) in microbes and their host can be detected by genomic and proteomic approaches. Genome-wide approaches to genotyping and phenotyping or expression profiling will eventually lead to global dissection of microbial pathogenesis, efficient and rapid diagnosis of infectious diseases, and the development of novel strategies to control infections. The key fundamental issue of infectious diseases is how to globally and integratively understand the interactions between microbial pathogens and their hosts by using infectomics. In this review, we focus on the events that are considered important in infectomics. Electronic Publication     

Point-of-care nucleic acid testing for infectious diseases

Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-based, can only be used in hospitals, and are still relatively complex and expensive. Integrating sample preparation with nucleic acid amplification and detection in a cost-effective, robust, and user-friendly format remains challenging. This review describes recent technical advances that might be able to address these limitations, with a focus on isothermal nucleic acid amplification methods. It briefly discusses selected applications related to the diagnosis and management of tuberculosis, HIV, and perinatal and nosocomial infections.     

Altered expression of adhesion molecules in inflammatory cervical smears

Objective:  The aim of the present study was to evaluate the expression of pan-cadherin and β-catenin in cervical smears with various types of infectious agents.
Patients and Methods:  Cervical smears obtained from 53 women, aged 21–65 years, with a diagnosis of specific inflammation were examined in our study. Eighteen subjects were infected by Candida albicans , 18 by Gardnerella vaginalis , nine by Bacteroides spp. and eight by Chlamydia trachomatis . All infectious agents found in the smears were at the same time confirmed by the microbiological laboratory methods. We performed a biotin–streptavidin-peroxidase immunocytochemical method using anti-β-catenin (Clone 12F7) and anti-pan-cadherin (pan, polyclonal) antibodies.
Results:  Aberrant expression of pan-cadherin was found in the cytoplasmic membrane of glandular, metaplastic, superficial and intermediate squamous cells in all types of infections. With regard to β-catenin, this was expressed in majority (90%) of glandular and metaplastic cells in all types of infections and in a small proportion (15%) of superficial and intermediate squamous cells in infections caused by C. albicans and G . vaginalis .
Conclusion:  Our data show that infectious agents may cause alterations in the expression and distribution of these adhesive molecules, which can be recognized in cervical smears. Additional studies in larger sets of patients should help clarify this issue further.     

Modeling the onset of symptoms of COVID-19: Effects of SARS-CoV-2 variant

Identifying order of symptom onset of infectious diseases might aid in differentiating symptomatic infections earlier in a population thereby enabling non-pharmaceutical interventions and reducing disease spread. Previously, we developed a mathematical model predicting the order of symptoms based on data from the initial outbreak of SARS-CoV-2 in China using symptom occurrence at diagnosis and found that the order of COVID-19 symptoms differed from that of other infectious diseases including influenza. Whether this order of COVID-19 symptoms holds in the USA under changing conditions is unclear. Here, we use modeling to predict the order of symptoms using data from both the initial outbreaks in China and in the USA. Whereas patients in China were more likely to have fever before cough and then nausea/vomiting before diarrhea, patients in the USA were more likely to have cough before fever and then diarrhea before nausea/vomiting. Given that the D614G SARS-CoV-2 variant that rapidly spread from Europe to predominate in the USA during the first wave of the outbreak was not present in the initial China outbreak, we hypothesized that this mutation might affect symptom order. Supporting this notion, we found that as SARS-CoV-2 in Japan shifted from the original Wuhan reference strain to the D614G variant, symptom order shifted to the USA pattern. Google Trends analyses supported these findings, while weather, age, and comorbidities did not affect our model’s predictions of symptom order. These findings indicate that symptom order can change with mutation in viral disease and raise the possibility that D614G variant is more transmissible because infected people are more likely to cough in public before being incapacitated with fever.