Bacterial diagnosisBacterial 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 LABORATORY DIAGNOSIS AND PROPHYLAXIS OF BACTERIAL INFECTIONOF BACTERIAL INFECTION , Treatment of Treatment of Bacterial InfectionBacterial Infection

Typical Culture Typical Culture Laboratory BenchLaboratory Bench

Koch‘s postulatesKoch‘s postulates

• IIsolated solated – diseased not healthy peoplediseased not healthy people

• GGrowthrowth– pure culturepure culture

• IInduce disease nduce disease – susceptible animalssusceptible animals

• RRe-isolated e-isolated – susceptible animalssusceptible 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

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.Step 1.

• SamplesSamples of body fluids (e.g. blood, urine, cerebrospinal of body fluids (e.g. blood, urine, cerebrospinal fluid) are inoculated on the plates and isolated colonies of fluid) are inoculated on the plates and isolated colonies of bacteria (which are visible to the naked eye) appear after bacteria (which are visible to the naked eye) appear after incubation for one - several days incubation for one - several days ..

• Each Each colonycolony consists of millions of bacterial cells. consists of millions of bacterial cells. Observation of these colonies for size, textureObservation of these colonies for size, texture 质地质地 , color, , color, and (if grown on blood agar) hemolysis reactions, is highly and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. important as a first step in bacterial identification.

• Whether the organism requires oxygen for growth is Whether the organism requires oxygen for growth is another important differentiating characteristic.another important differentiating characteristic.

Step 2.Step 2.• Colonies are Gram stained and individual bacterial Colonies are Gram stained and individual bacterial

cells observed under the cells observed under the microscope.microscope.

DIFFERENTIATION OF BACTERIAGRAM STAINGRAM STAIN

Gram NegativeGram Positive

To rapid diagnosisTo rapid diagnosis• DDirect microscopicirect microscopic observation of certain clinical observation of certain clinical

samples for the presence of bacteria can be helpful samples for the presence of bacteria can be helpful (e.g. detection of (e.g. detection of M. tuberculosisM. tuberculosis in sputum). in sputum).

a cb

Approaches to rapid diagnosis Approaches to rapid diagnosis without prior culturewithout prior culture• Certain human pathogens (including the causative Certain human pathogens (including the causative

agents of tuberculosis, Lyme disease and syphilisagents of tuberculosis, Lyme disease and syphilis梅毒梅毒 ) either cannot be isolated in the laboratory or ) either cannot be isolated in the laboratory or grow extremely poorly. Successful isolation can be grow extremely poorly. Successful isolation can be slow and in some instances impossible. Direct slow and in some instances impossible. Direct detection of bacteria without culture is possible in detection of bacteria without culture is possible in some cases.some cases.

COLONIAL MORPHOLOGY

固体培养基：菌落固体培养基：菌落 colonycolony ，菌苔，菌苔

Blood Agar PlateBlood Agar Plate

Step 3Step 3

• The bacteria are speciated using these isolated The bacteria are speciated using these isolated colonies. This often requires an additional 24 hr of colonies. This often requires an additional 24 hr of growth. growth.

• IdentificationIdentification: biochemical reactions; immunological : biochemical reactions; immunological method (using known antisera); drug sensitivity test; method (using known antisera); drug sensitivity test; etc. 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 diagnosisA simple approach to rapid diagnosis-1-1

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

SSimple approach to rapid diagnosisimple approach to rapid diagnosis-2-2

• Serologic identificationSerologic identification of an antibody response (in of an antibody response (in patient's serum) to the infecting agent can only be patient's serum) to the infecting agent can only be successful several weeks after an infection has successful several weeks after an infection has occurred.occurred.

• include agglutination,precipitation, complement include agglutination,precipitation, complement fixation test,neutralization, etc.fixation test,neutralization, etc.

AGGLUTINATION IMMUNOFLUORESCENCE

ANTIGENIC STRUCTURE

Widal testWidal test

Serological agglutinated test

PATHOGENICITY :animal test model

Such as Susceptibility of Experimental host to clinical Isolate

By ELASA tesaBy ELASA tesa to rapid diagnosis to rapid diagnosis

By molecular biologicalBy molecular biological skills to rapid skills to rapid diagnosisdiagnosis

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

获得性免疫产生的方式获得性免疫产生的方式• 主动免疫 自然免疫 主动免疫 自然免疫 患病、隐性感染患病、隐性感染 人工免疫 人工免疫 疫苗、类毒素疫苗、类毒素 • 被动免疫 自然免疫 被动免疫 自然免疫 胎盘、初乳胎盘、初乳 人工免疫 人工免疫 抗毒素、丙球抗毒素、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 生物制品：人工免疫的免疫原、免疫血清、细胞制剂及诊断 制剂的生物性制剂制剂的生物性制剂

Artificial active Artificial active immunizationimmunization

• Inactivated vaccineInactivated vaccine

• Attenuated VaccineAttenuated Vaccine

• ToxoidToxoid

• AutovaccineAutovaccine

• Polysaccharide vaccine Polysaccharide vaccine

Artificial active Artificial active immunizationimmunization

• Chemical vaccineChemical vaccine

• Synthetic VaccineSynthetic Vaccine

• Gene-defective vaccineGene-defective vaccine

• DNA recombinant vaccineDNA 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 Artificial passive immunizationimmunization

• AntitoxinAntitoxin

• Antibacterial antibodyAntibacterial antibody

• Placental globulinPlacental globulin

• Gamma globulinGamma globulin

• TFTF

• IFNIFN

• 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）

区别要点区别要点 人工主动免疫人工主动免疫 人工被动免疫人工被动免疫

免疫物质免疫物质 抗原抗原 抗体或细胞因子等抗体或细胞因子等

免疫出现时间免疫出现时间 慢，慢， 2~42~4周周 快，立即快，立即

免疫维持时间免疫维持时间 长，数年长，数年 ~~ 数月数月 短，短， 2~32~3周周

主要用途主要用途 预防预防 治疗或紧急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 Treatment