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Laboratory Diagnosis, Prevention and Treatment of Bacterial Infection • Manifestations of Infection: Signs and symptoms vary according to the site and severity of infection. Diagnosis requires a composite of information, including history, physical examination, radiographic findings, and laboratory data. • Microbial Causes of Infection: Infections may be caused by bacteria, viruses, fungi, and parasites. The pathogen may be exogenous (acquired from environmental or animal sources or from other persons) or endogenous (from the normal flora). Specimen Selection, Collection, and Processing • The quantity material must be adequate • Specimens are selected on the basis of signs and symptoms, should be representative of the disease process • Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions • The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful. • Meaningful specimens to diagnose bacterial infections must be secured before antimicrobial drugs are administered. Bacteria Identification • Since the discovery of microorganisms a few centuries ago, scientists have been looking for better ways to identify bacteria. • Some methods are old and time tested, while some methods are new and genetically based. • Either way, a very specific work flow is required to identify bacteria. Microscopic Examination Microscopic examination is usually done by way of the gram stain procedure. This staining method will differentiate gram-positive (purple) bacteria from gram-negative (pink) bacteria. This is based on the composition of the cell wall of the organism. Common gram-positive bactera are staphylococcus and streptococcus. Gram-negative bacteria include E. coli and Klebsiella. In addition, microscopic morphology that is useful in identification can be noted, for example staphylococci are gram-positive cocci in clusters. So the gram stain appears microscopically as purple spheres in a formation resembling a group of grapes. Media Selection Based on the specimen source (urine, blood, etc.) and the likely organisms that would be present, the appropriate bacteriological media are selected to grow the organism for further work-up. In addition, selecting the appropriate temperature and atmospheric conditions (aerobic versus anaerobic) will allow bacterial growth. Colony Morphology How the bacterial colony appears on bacteriological growth media offers key characteristics about the bacteria. Characteristics like size, color, odor and the ability to lyse (break apart) blood cells are all important identification criteria for determining what the unknown bacteria is. Biochemical and Enzymatic Tests The ability of a bacterial species to use a biochemical, a sugar, amino acid or a enzymatic substrate is very useful in identifying bacteria. These tests can be used individually (coagulase for Staphylococcus aureus) to identify a organism, or in a battery of tests to identify gram-negative bacilli. Many commercially prepared tests kits are available to identify bacteria using biochemicals and enzymes. Serological Methods Serologic methods for identifying bacteria are frequently used in the laboratory. This typically involves testing an unknown antibody against a known antigen bound to a latex particle or similar structure. After mixing the two together and rotating, a visible agglutination (clumping) will appear if positive for the organism tested. Other tests look for antibody neutralization, inhibition and other end products. Molecular Methods The latest and most specific methods available in the laboratory are molecular methods. These tests are based on the ability to detect, identify and characterize microorganisms based on their DNA or RNA. Polymerase chain reaction (PCR) is a common molecular method used to identify bacteria. Microbiologic Examination • Culture:Isolation of infectious agents frequently requires specialized media. Nonselective (noninhibitory) media permit the growth of many microorganisms. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms. • Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic probes are also used. Microbiologic Examination • Antimicrobial Susceptibility: Microorganisms, particularly bacteria, are tested in vitro to determine whether they are susceptible to antimicrobial agents. • Serodiagnosis:A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis. • Direct Examination and Techniques: Direct examination of specimens reveals gross pathology. Microscopy may identify microorganisms. Immunofluorescence, immuno-peroxidase staining, and other immunoassays may detect specific microbial antigens. Genetic probes identify genus- or species-specific DNA or RNA sequences. Artificial active immunity Vaccines are antigens prepared from pathogens that can raise a protective immune response, yet do not cause illness. These prepared antigens will stimulate both B cells and T cells and help to create memory cells that can later mount a vigorous immune response to an encounter with the real pathogen. • Toxoids: a modified form of the toxin that preserves its antigenicity but has lost its toxicity. This has been spectacularly successful with tetanus and diphtheria. Artificial active immunity • Inactivated vaccines: The production of protective antibodies is stimulated by using the killed (inactivated) organisms This is done as a routine with vaccines against pertussis (whooping cough) , typhoid and influenza. There is also an inactivated polio vaccine. • Attenuated live vaccines : The approach is to use suspensions of living organisms that are reduced in their virulence (attenuated) but still immunogenic. This strategy has yielded: BCG, mumps, measles , and rubella vaccines (now combined); the live virus polio vaccine. • Special vaccines: polysaccharide vaccine, subunit vaccine, ( conjugate vaccine, bio-engineered vaccine, chemical vaccine, synthetic vaccine ), nucleic acid vaccine, idiotype vaccine, autovaccine, etc. Artificial passive immunity • • • • Antitoxin: e.g. Tetanus antitoxin and diphtheria antitoxin. It is raised in the horse .It is most important to give an intented recipient of equine serum a prior test dose to exclude hypersensitivity subjects who may have been sensitized by a previous dose of equine serum. Pooled immunoglobulin: It contains the normal repertoire of antibodies for an adult, and can protect against hepatitis A, and measles. Specific immunoglobulin: Preparations of specific immunoglobulin are available for passive immunization against tetanus, hepatitis B, rabies, varicella-zoster. Cytokine Active-passive immunity • involves giving both a vaccine to provide long-term protection (preventive infection) and immune globulin to provide immediate protection (therapeutic and preventive infectious disease). 人工主动免疫与人工被动免疫的区别 区别要点 人工主动免疫 人工被动免疫 免疫物质 抗原 抗体或细胞因子等 免疫出现时间 慢，2~4周 快，立即 免疫维持时间 长，数年~数月 短，2~3周 主要用途 治疗或紧急预防 预防 General procedure for collecting and processing specimens for aerobic and/or anaerobic bacterial culture Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.