Causation and disease: effect of technology on postulates of causation.

This paper reviews the technical developments in microbiology that led to the discovery of new infectious agents and the effect of these discoveries on establishing proof of causation. In bacteriology, these advances included the light microscope, bacterial stains, bacterial cultures, and the methods used to isolate clones. In virology, they involved the use of filters to separate viruses from bacteria, the electron microscope, the use of laboratory animals, embryonated eggs, tissue cultures to identify or grow the agent, and the recent development of molecular techniques to detect the presence of antigen in tissues. In immunology, they were based on the discovery of antibodies and of the immune response.     

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.     

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.     

Enzyme immunoassay and polymerase chain reaction for evaluation of brucella persistence

The complex approach, including the use of traditional bacteriological and serological methods, as well as the polymerase chain (PCR) reaction and the enzyme immunoassay (EIA), was used for evaluation of Brucella (the causative agents of brucellosis) persistence in the dynamics of the infectious process in patients with the acute and chronic forms of brucellosis as well as in experimentally infected laboratory animals. Sick humans and experimental animals were found to have positive PCR and EIA reactions at different periods of the disease. The use of these methods makes it possible to evaluate indirectly the persistence of Brucella.     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.     

Molecular-genetic approaches to diagnosis and intraspecific typing of causative agents of glanders and melioidosis

Pathogenic Burkholderia--Burkholderia mallei and Burkholderia pseudomallei--are causative agents of glanders and melioidosis, severe infectious diseases of man and animals. They are regarded as potential agents of bioterrorism. The existing bacteriological and immunological methods of identification of B. mallei and B. pseudomallei are not efficient enough for the rapid diagnosis and typing of strains. Described in the paper are molecular methods of detection of the agents by PCR, hybridization and strain typing made on the basis of bacterial total cell protein profiles, RAPD, ribotyping as well as of plasmid and DNA microrestriction analyses.     

Lurking in the shadows: emerging rodent infectious diseases

Rodent parvoviruses, Helicobacter spp., murine norovirus, and several other previously unknown infectious agents have emerged in laboratory rodents relatively recently. These agents have been discovered serendipitously or through active investigation of atypical serology results, cell culture contamination, unexpected histopathology, or previously unrecognized clinical disease syndromes. The potential research impact of these agents is not fully known. Infected rodents have demonstrated immunomodulation, tumor suppression, clinical disease (particularly in immunodeficient rodents), and histopathology. Perturbations of organismal and cellular physiology also likely occur. These agents posed unique challenges to laboratory animal resource programs once discovered; it was necessary to develop specific diagnostic assays and an understanding of their epidemiology and transmission routes before attempting eradication, and then evaluate eradication methods for efficacy. Even then management approaches varied significantly, from apathy to total exclusion, and such inconsistency has hindered the sharing and transfer of rodents among institutions, particularly for genetically modified rodent models that may not be readily available. As additional infectious agents are discovered in laboratory rodents in coming years, much of what researchers have learned from experiences with the recently identified pathogens will be applicable. This article provides an overview of the discovery, detection, and research impact of infectious agents recently identified in laboratory rodents. We also discuss emerging syndromes for which there is a suspected infectious etiology, and the unique challenges of managing newly emerging infectious agents.     

Conventional methods and future trends in antimicrobial susceptibility testing

Antimicrobial susceptibility testing is an essential task for selecting appropriate antimicrobial agents to treat infectious diseases. Constant evolution has been observed in methods used in the diagnostic microbiology laboratories. Disc diffusion or broth microdilution are classical and conventional phenotypic methods with long turnaround time and labour-intensive but still widely practiced as gold-standard. Scientists are striving to develop innovative, novel and faster methods of antimicrobial susceptibility testing to be applicable for routine microbiological laboratory practice and research. To meet the requirements, there is an increasing trend towards automation, genotypic and micro/nano technology-based innovations. Automation in detection systems and integration of computers for online data analysis and data sharing are giant leaps towards versatile nature of automated methods currently in use. Genotypic methods detect a specific genetic marker associated with resistant phenotypes using molecular amplification techniques and genome sequencing. Microfluidics and microdroplets are recent addition in the continuous advancement of methods that show great promises with regards to safety and speed and have the prospect to identify and monitor resistance mechanisms. Although genotypic and microfluidics methods have many exciting features, however, their applications into routine clinical laboratory practice warrant extensive validation. The main impetus behind the evolution of methods in antimicrobial susceptibility testing is to shorten the overall turnaround time in obtaining the results and to enhance the ease of sample processing. This comprehensive narrative review summarises major conventional phenotypic methods and automated systems currently in use, and highlights principles of some of the emerging genotypic and micro/nanotechnology-based methods in antimicrobial susceptibility testing.     

Detection of infectious prions in urine

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.     

Infectious animal disease and its control

The control of infectious diseases in the main food-producing animals is considered and the main factors involved in the epizootiology of disease are presented. The properties of infectious agents and their natural history together with factors that influence the spread and development of disease are summarized. The factors in intensive animal husbandry that affect the occurrence of infectious disease and its control are considered. These include population density, population movement, management, hygiene and genetic constitution of the host. They encourage the appearance of new diseases, changes in the character of established diseases and the development of pathogenicity in infectious agents that were previously of no importance. Intensive animal husbandry has also increased the importance of multifactorial disease, which includes diseases that require more than one infectious agent or one or more infectious agents plus other factors for their cause. The methods of control of infectious disease currently available are described and the success and difficulties of their control on a global, national and local (farm or enterprise) basis are considered. Examples of diseases of global importance where national and world programmes of control and eradication have been of varying success are described. Examples of diseases that are enzootic throughout the world and the procedures used for their control are also described. The technological opportunities for the improvement of the control of infectious disease in the future are discussed. It is considered that developments in molecular biology and immunology will provide improvements in diagnostic tools and will revolutionize the development of animal resistance to disease and the production and use of vaccines.     

Protein misfolding cyclic amplification of infectious prions

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP(C) and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP(Sc) and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.     

Diagnosis of Dermatophytosis Using Molecular Biology

Identification of fungi in dermatological samples using PCR is reliable and provides significantly improved results in comparison with cultures. It is possible to identify the infectious agent when negative results are obtained from cultures. In addition, identification of the infectious agent can be obtained in 1 day. Conventional and real-time PCR methods used for direct fungus identification in collected samples vary by DNA extraction methods, targeted DNA and primers, and the way of analysing the PCR products. The choice of a unique method in a laboratory is complicated because the results expected from skin and hair sample analysis are different from those expected in cases of onychomycosis. In skin and hair samples, one dermatophyte among about a dozen possible species has to be identified. In onychomycosis, the infectious agents are mainly Trichophyton rubrum and, to a lesser extent, Trichophyton interdigitale, but also moulds insensitive to oral treatments used for dermatophytes, which renders fungal identification mandatory. The benefits obtained with the use of PCR methods for routine analysis of dermatological samples have to be put in balance with the relative importance of getting a result in a short time, the price of molecular biology reagents and equipment, and especially the time spent conducting laboratory manipulations.     

Molecular diagnostics of medically important bacterial infections

Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world's biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. In developed countries, the emergence of new, rare or already-forgotten infectious diseases, such as HIV/AIDS, Lyme disease and tuberculosis, has stimulated public interest and inspired commitments to surveillance and control. Recently, it is reported that infectious diseases are responsible for more than 17 million deaths worldwide each year, most of which are associated with bacterial infections. Hence, the control of infectious diseases control is still an important task in the world. The ability to control such bacterial infections is largely dependent on the ability to detect these aetiological agents in the clinical microbiology laboratory. Diagnostic medical bacteriology consists of two main components namely identification and typing. Molecular biology has the potential to revolutionise the way in which diagnostic tests are delivered in order to optimise care of the infected patient, whether they occur in hospital or in the community. Since the discovery of PCR in the late 1980s, there has been an enormous amount of research performed which has enabled the introduction of molecular tests to several areas of routine clinical microbiology. Molecular biology techniques continue to evolve rapidly, so it has been problematic for many laboratories to decide upon which test to introduce before that technology becomes outdated. However the vast majority of diagnostic clinical bacteriology laboratories do not currently employ any form of molecular diagnostics but the use such technology is becoming more widespread in both specialized regional laboratories as well as in national reference laboratories. Presently molecular biology offers a wide repertoire of techniques and permutations of these analytical tools, hence this article wishes to explore the application of these in the diagnostic laboratory setting.     

Epidemiology and disease control in everyday beef practice

It is important for food animal veterinarians to understand the interaction among animals, pathogens, and the environment, in order to implement herd-specific biosecurity plans. Animal factors such as the number of immunologically protected individuals influence the number of individuals that a potential pathogen is able to infect, as well as the speed of spread through a population. Pathogens differ in their virulence and contagiousness. In addition, pathogens have various methods of transmission that impact how they interact with a host population. A cattle population's environment includes its housing type, animal density, air quality, and exposure to mud or dust and other health antagonists such as parasites and stress; these environmental factors influence the innate immunity of a herd by their impact on immunosuppression. In addition, a herd's environment also dictates the "animal flow" or contact and mixing patterns of potentially infectious and susceptible animals. Biosecurity is the attempt to keep infectious agents away from a herd, state, or country, and to control the spread of infectious agents within a herd. Infectious agents (bacteria, viruses, or parasites) alone are seldom able to cause disease in cattle without contributing factors from other infectious agents and/or the cattle's environment. Therefore to develop biosecurity plans for infectious disease in cattle, veterinarians must consider the pathogen, as well as environmental and animal factors.     

The use of polymerase chain reaction for detection of the agents of glanders and melioidosis using experimental infection

Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material.     

羊瘙痒因子263K毒株感染金黄地鼠后鼠脑组织中检出PrP-res蛋白及其神经病理学研究

羊瘙痒病是累及山羊及绵羊的可传播海绵状脑病。为了观察羊瘙痒因子 (Scrapie)的病原特征及病理组织改变特点 ,将羊瘙痒因子 2 6 3K毒株颅内接种至金黄地鼠。经过 81～ 110天的潜伏期 ,89%的动物发病 (17/19只 )。对发病地鼠的神经病理学检测发现 ,海绵状空泡变性的检出率为 5 9% ,淀粉样斑的检出率为 17 6 %。利用免疫组化和蛋白酶消化后的Westernblotting检测证实 ,10 0 %的发病地鼠的脑组织中都出现蛋白酶抗性朊蛋白 (PrP res)。17只发病地鼠脑组织提取物中 ,PrP res的泳动位置和分子量大小完全一致 ,出现两条分子量在 2 5kD～ 31kD的反应带。尝试应用快速玻片印迹法检测病变组织中的PrP res,结果显示 ,与常规固定包埋切片的免疫组化检出效果相似。这提示脑组织印片法可成为临床检测克 雅氏病 (Creutzfeldt Jacobdisease ,CJD)患者脑组织活检标本中PrP res的快速、有效的方法。羊瘙痒因子 2 6 3K成功感染金黄地鼠再次证明 ,金黄地鼠是TSE感染因子良好的动物模型 ,发病率高 ,潜伏期短 ,发病动物PrP res的检出率明显高于典型病理改变的检出率。新生成的PrP res的电泳类型与接种的TSE因子有关 ,与宿主的个体差异无关 ,提示TSE感染因子的确存在“株”的现象。     

Eritema multiforme secundario a infección por Trichophyton mentagrophytes

Erythema multiforme is an acute self-limited cutaneous syndrome associated in more than 50% of the cases with herpes simplex virus infection; but it can also be a consequence of other infectious agents or drugs. We report on a 24 year-old female patient with erythema multiforme secondary to Trichophyton mentagrophytes var. mentagrophytes cutaneous infection acquired from contact with animals in a research laboratory.     

Viral battery testing in nonhuman primate colony management

Good colony management is associated with monitoring of animals for infectious agents. Of major current concern are B virus and simian AIDS (SAIDS) viruses. However, other viral agents frequently cause serious disease outbreaks which can be avoided if their presence is detected sufficiently early. The recent development of a rapid, sensitive and specific diagnostic test system, i.e., the dot immunobinding assay (DIA) permits the monitoring of a colony for many of the viruses that pose problems. By employing battery type testing using a panel of appropriate viral antigens, investigators are able to detect the increased presence of viral agents of concern and take necessary measures to prevent extension of the problem.     

Synthetic peptide vaccines

The review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modeling of human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. The review contains examples of the developments of synthetic peptide vaccines against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease).     

Pathobiology and management of laboratory rodents administered CDC category A agents

The Centers for Disease Control and Prevention Category A infectious agents include Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), variola major virus (smallpox), Francisella tularensis (tularemia), and the filoviruses and arenaviruses that induce viral hemorrhagic fevers. These agents are regarded as having the greatest potential for adverse impact on public health and therefore are a focus of renewed attention in infectious disease research. Frequently rodent models are used to study the pathobiology of these agents. Although much is known regarding naturally occurring infections in humans, less is documented on the sources of exposures and potential risks of infection to researchers and animal care personnel after the administration of these hazardous substances to laboratory animals. Failure to appropriately manage the animals can result both in the creation of workplace hazards if human exposures occur and in disruption of the research if unintended animal exposures occur. Here we review representative Category A agents, with a focus on comparing the biologic effects in naturally infected humans and rodent models and on considerations specific to the management of infected rodent subjects. The information reviewed for each agent has been curated manually and stored in a unique Internet-based database system called HazARD (Hazards in Animal Research Database, http://helab.bioinformatics.med.umich.edu/hazard/) that is designed to assist researchers, administrators, safety officials, Institutional Biosafety Committees, and veterinary personnel seeking information on the management of risks associated with animal studies involving hazardous substances.