Bacterial diagnosis Principle of collection sample and sending To avoid contaminants Collect sample time Before using antibiotics Sample come from main infectious site To take notice of conservation in tranport The sample mast be fresh LABORATORY DIAGNOSIS AND PROPHYLAXIS OF BACTERIAL INFECTION Treatment of Bacterial Infection LABORATORY DIAGNOSIS AND PROPHYLAXIS OF BACTERIAL INFECTION, Treatment of Bacterial Infection

Typical Culture Laboratory Bench

Koch ‘ s postulates Isolated Isolated –diseased not healthy people Growth Growth –pure culture Induce disease Induce disease –susceptible animals Re-isolated Re-isolated –susceptible animals

致病菌的检验程序 Drug sensitivity 药物敏感试验 Directing smear exam Isolated culture 分离培养 Biochemistry essay 生化试验 Serologic test 血清学试验 Animal test 动物试验 明确诊断明确诊断 First diagnosis Molecular biologic skill 分子生物学技术 Else detect skill 其他检测技术

A. CINICALLY BY THE TYPE OF DISEASE

Step 1. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are inoculated on the plates and isolated colonies of bacteria (which are visible to the naked eye) appear after incubation for one - several days. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are inoculated on the plates and isolated colonies of bacteria (which are visible to the naked eye) appear after incubation for one - several days. Each colony consists of millions of bacterial cells. Observation of these colonies for size, texture 质地, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. Each colony consists of millions of bacterial cells. Observation of these colonies for size, texture 质地, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. Whether the organism requires oxygen for growth is another important differentiating characteristic. Whether the organism requires oxygen for growth is another important differentiating characteristic.

Step 2. Colonies are Gram stained and individual bacterial cells observed under the microscope. Colonies are Gram stained and individual bacterial cells observed under the microscope.

DIFFERENTIATION OF BACTERIA GRAM STAIN Gram NegativeGram Positive

To rapid diagnosis Direct microscopic observation of certain clinical samples for the presence of bacteria can be helpful (e.g. detection of M. tuberculosis in sputum). Direct microscopic observation of certain clinical samples for the presence of bacteria can be helpful (e.g. detection of M. tuberculosis in sputum).

acb

Approaches to rapid diagnosis without prior culture Certain human pathogens (including the causative agents of tuberculosis, Lyme disease and syphilis 梅毒 ) either cannot be isolated in the laboratory or grow extremely poorly. Successful isolation can be slow and in some instances impossible. Direct detection of bacteria without culture is possible in some cases. Certain human pathogens (including the causative agents of tuberculosis, Lyme disease and syphilis 梅毒 ) either cannot be isolated in the laboratory or grow extremely poorly. Successful isolation can be slow and in some instances impossible. Direct detection of bacteria without culture is possible in some cases.

COLONIAL MORPHOLOGY

固体培养基：菌落 colony ，菌苔 Blood Agar Plate

Step 3 The bacteria are speciated using these isolated colonies. This often requires an additional 24 hr of growth. The bacteria are speciated using these isolated colonies. This often requires an additional 24 hr of growth. Identification: biochemical reactions; immunological method (using known antisera); drug sensitivity test; etc. Identification: biochemical reactions; immunological method (using known antisera); drug sensitivity test; etc.

Bacteria Biochemistry test

原理 血清学诊断 双份血清标本，恢复期血清中抗体效价比急性期 血清中抗体效价升高 ≥4 倍者方有意义 种类 用已知的细菌或其特异性抗原检测患者体 液中有无相应特异性抗体和其效价的动态变化， 可作为某些传染病的辅助诊断。 ( 抗原 → 未知抗体 ) 标本 凝集试验： 沉淀试验： 补体结合试验： 中和试验： 肥达试验、外斐试验、显微镜凝集试验等 梅毒 VDRL 、 RPR 试验等 抗 “O” 试验等 Q 热柯克斯体抗体检测等

A simple approach to rapid diagnosis-1 antigen detection is used in many doctor‘s offices for the group A streptococcus. The patient’s throat is swabbed and streptococcal antigen extracted directly from the swab (without prior bacteriological culture). The bacterial antigen is detected by aggregation (agglutination) of antibody coated latex beads 乳胶微球. antigen detection is used in many doctor‘s offices for the group A streptococcus. The patient’s throat is swabbed and streptococcal antigen extracted directly from the swab (without prior bacteriological culture). The bacterial antigen is detected by aggregation (agglutination) of antibody coated latex beads 乳胶微球.

Simple approach to rapid diagnosis-2 Serologic identification of an antibody response (in patient's serum) to the infecting agent can only be successful several weeks after an infection has occurred. Serologic identification of an antibody response (in patient's serum) to the infecting agent can only be successful several weeks after an infection has occurred. include agglutination,precipitation, complement fixation test,neutralization, etc. include agglutination,precipitation, complement fixation test,neutralization, etc.

AGGLUTINATIONIMMUNOFLUORESCENCE ANTIGENIC STRUCTURE

Widal test Serological agglutinated test

PATHOGENICITY :animal test model Such as Susceptibility of Experimental host to clinical Isolate

By ELASA tesa to rapid diagnosis

By molecular biological skills to rapid diagnosis Bacterial DNA sequences can be amplified directly from human body fluids (the polymerase chain reaction, PCR). In this fashion large amounts of specific genes or portions of genes can be generated and readily detected. For example, great success has been achieved in rapid diagnosis of tuberculosis. Bacterial DNA sequences can be amplified directly from human body fluids (the polymerase chain reaction, PCR). In this fashion large amounts of specific genes or portions of genes can be generated and readily detected. For example, great success has been achieved in rapid diagnosis of tuberculosis.

MOLECULAR-GENETIC METHODS USING RNA AND DNA SEQUENCES AS SPECIFIC PROBES

DNA HOMOLOGY AND G/C RATIOS (GENOMICS- DNA SEQUENCING)

DNA RESTRICTION LENGTH POLYMORPHISMS (RFLP) AND POLYMERASE CHAIN REACTIONS (PCR)

PHAGE SUSCEPTABILITY AND PHAGE TYPING Diagram of a representative bacteriophage Phage typing is used primarily to differentiate groups within Staphylococcus and Enterobacteriaceae

获得性免疫产生的方式 主动免疫 自然免疫 患病、隐性感染 主动免疫 自然免疫 患病、隐性感染 人工免疫 疫苗、类毒素 人工免疫 疫苗、类毒素 被动免疫 自然免疫 胎盘、初乳 被动免疫 自然免疫 胎盘、初乳 人工免疫 抗毒素、丙球 人工免疫 抗毒素、丙球 人工免疫：采用人工方法将疫苗、类毒素等或含有某种特异 性抗体、细胞免疫制剂等接种于人体，以增强宿主体的抗 病能力 生物制品：人工免疫的免疫原、免疫血清、细胞制剂及诊断 制剂的生物性制剂

Artificial active immunization Inactivated vaccine Inactivated vaccine Attenuated Vaccine Attenuated Vaccine Toxoid Toxoid Autovaccine Autovaccine Polysaccharide vaccine Polysaccharide vaccine

Artificial active immunization Chemical vaccine Chemical vaccine Synthetic Vaccine Synthetic Vaccine Gene-defective vaccine Gene-defective vaccine DNA recombinant vaccine DNA recombinant vaccine Idiotype vaccine Idiotype vaccine

将疫苗或类毒素接种于人体，使机体产生获 得性免疫力的一种防治微生物感染的措施。 作用：预防 人工主动免疫 artificial active immunity 主要生物制品  死疫苗  活疫苗  类毒素  亚单位疫苗  DNA 重组疫苗  核酸疫苗  治疗性疫苗

区别点死疫苗活疫苗 制剂特点死，强毒株活减毒株 接种剂量与特点量较大， 2~3 次 量较小， 1 次 保存及有效期易保存，约 1 年不易保存 免疫效果较低， 维持数月 ~2 年 较高， 维持 3~5 年甚至更长 死疫苗与活疫苗的区别

Artificial passive immunization Antitoxin Antitoxin Antibacterial antibody Antibacterial antibody Placental globulin Placental globulin Gamma globulin Gamma globulin TF TF IFN IFN IL-2 IL-2

抗毒素 人工被动免疫 artificial passive immunity 注射含有特异性抗体的免疫血清或纯化免 疫球蛋白抗体，或细胞因子等免疫制剂，使机 体即刻获得特异性免疫，作用及时。 作用：治疗，紧急预防 主要生物制品 丙种球蛋白 ( 麻疹、甲肝、脊髓灰质炎）

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 主要用途预防治疗或紧急预防 人工主动免疫与人工被动免疫的区别

Treatment