Laboratory Diagnosis of Viral diseases LONG JIAN-ER, PhE Lab of Medical Microbiology, Department of Medical Microbiology and Parasitology, Shang Medical College of Fudan University, 138 Yixueyuanr, shanghai 200032, pr china Email:longjianer@fudan.edu.cn;Tel:+86-21-54237867
Laboratory Diagnosis of Viral Diseases LONG JIAN-ER, Ph.D. Lab of Medical Microbiology, Department of Medical Microbiology and Parasitology, Shang Medical College of Fudan University, 138 Yixueyuan R., Shanghai 200032, PR China Email: longjianer@fudan.edu.cn; Tel.: +86-21-54237867
Kochs Postulates Organism present only in diseased individuals 2. Organism cultivated in pure culture from diseased individual KOCHS POSTULATES Diseased Healthy The Postulates 1.The suspected pathogenic Microscopy, staining organism should be present in all cases of the blood/tissue disease and absent from healthy animals Suspected microscope 2. The suspected organismLaboratory culture treak agar plate should be grown in pure suspected healthy animal Inoculate healthy anima cells of suspected patho
Koch’s Postulates 1. Organism present only in diseased individuals 2. Organism cultivated in pure culture from diseased individual
Koch’ s Postu|ates 3. Organism causes disease when injected into healthy individuals 4. Organism re-isolated from infected individual from point 3 KOCHS POSTULATES Diseased The postulates: Tools: 3. Cells from a pure culture Experimental animal of the suspected organism should cause disease in a Diseased animal healthy animal Remove blood or tissue sample and observe by microscopy 4. The organism should be aboratory reisolation Suspecte Laboratory Pure culture reisolated and shown to be (must b the same as the original
3. Organism causes disease when injected into healthy individuals 4. Organism re-isolated from infected individual from point 3. Koch’s Postulates
River’ s Postu|ates T.M. River, 1937 Modified from Koch's Postulates(proof of bacterial diseases) 1. Isolate virus from diseased hosts Cultivation of virus in host cells 3. Proof of filterability 4. Production of a comparable disease when the cultivated virus is used to infect experimental animals 5. Re-isolation of the same virus from the infected experimental animal 6. Detection of a specific immune response to the virus
River’s Postulates T.M. River, 1937 Modified from Koch’s Postulates (proof of bacterial diseases) 1. Isolate virus from diseased hosts. 2. Cultivation of virus in host cells. 3. Proof of filterability. 4. Production of a comparable disease when the cultivated virus is used to infect experimental animals. 5. Re-isolation of the same virus from the infected experimental animal. 6. Detection of a specific immune response to the virus
Procedures for laboratory viral diagnosis Virus infected patients Check the infected cells Observe virus under light microscope Collected specimens particles under electronic microscope Virus culture and Detection of viral antigen Serology test isolation or genome antibody test Identify viral propagation Antigen Genome Neutralization test Hemagglutination CPE inhibition test Hemadsorption Immunofluoresence PCR EIA/ELISA Hemagglutination EIAELISA Southern blot Western Blot Virus interference Western Blot Northern blot Neutralization test Immunoelectrophesis Dot blot Plaque assay Radio immune assay In situ hybridization TCiD50 assay
Virus infected patients Collected specimens Check the infected cells under light microscope Observe virus particles under electronic microscope Virus culture and isolation Serology test antibody test Detection of viral antigen or genome Identify viral propagation Antigen Genome Neutralization test Hemagglutination inhibition test EIA/ELISA Western Blot Immunofluoresence EIA/ELISA Western Blot Immunoelectrophesis Radio immune assay PCR Southern blot Northern blot Dot blot In situ hybridization CPE Hemadsorption Hemagglutination Virus interference Neutralization test Plaque assay TCID50 assay Procedures for laboratory viral diagnosis
Viral Diagnostics in the Clinical Laboratory a Over 70% of all infectious disease cases seen by a physician are due to viral infections a Quality of patient specimens and their transport to the laboratory is important
Viral Diagnostics in the Clinical Laboratory Over 70% of all infectious disease cases seen by a physician are due to viral infections. Quality of patient specimens and their transport to the laboratory is important
Relation of stage of illness to presence of virus in test material &s to appearance of specific antibody Stage or period Virus Specific of illness detectable antibody Incubation R arely No Prodrome Occasionally No Onset Frequently Occasionally Acute pl ase Frequently Frequently Recovery Rare Usually Convalescence ry rarely sually
Relation of stage of illness to presence of virus in test material & to appearance of specific antibody Stage or period of illness Virus detectable Specific antibody Incubation Rarely No Prodrome Occasionally No Onset Frequently Occasionally Acute phase Frequently Frequently Recovery Rarely Usually Convalescence Very rarely Usually
Storage and Collection of Biological Specimens for Viral Testing a What types of specimens are collected to diagnose? O Respiratory tract infections: Nasal and bronchial washings throat and nasal swabs, sputum O Eye infections. throat and conjunctival swab/scraping D Gastrointestinal tract infections: stool and rectal swabs a Vesicular rash: vesicle fluid, skin scrapings a Maculopapular rash: throat stool and rectal swabs O CNS(encephalitis and meningitis cases) stool, tissue, saliva, brain biopsy cerebrospinal fluid D Genital infections vesicle fluid or swab a Urinary tract infections: urine a Bloodborne infections: blood
Storage and Collection of Biological Specimens for Viral Testing What types of specimens are collected to diagnose? Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum Eye infections: throat and conjunctival swab/scraping Gastrointestinal tract infections: stool and rectal swabs Vesicular rash: vesicle fluid, skin scrapings Maculopapular rash: throat, stool, and rectal swabs CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid Genital infections: vesicle fluid or swab Urinary tract infections: urine Bloodborne infections: blood
Collection of vesicular fluid from palmar lesions for virological diagnosis of HFMD
Collection of vesicular fluid from palmar lesions for virological diagnosis of HFMD
Three General Approaches for Laboratory Diagnosis of Viral Infections ■ Direct detection a Microscopy or staining a Detection of nucleic acid, antigens Virus Isolation(Indirect Examination) D CPE and other characters nucleic acid antigens for detection ■ Serology □ Antibodies
Three General Approaches for Laboratory Diagnosis of Viral Infections Direct detection Microscopy or staining Detection of nucleic acid, antigens Virus Isolation (Indirect Examination) CPE and other characters nucleic acid, antigens for detection Serology Antibodies