26 - Laboratory Diagnosis 1 Category of Sample • Blood...

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Unformatted text preview: Laboratory Diagnosis 1 Category of Sample • Blood, Urine, Stool, nasal washing, nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab), tissue ,brain biopsy, cerebrospinal fluid, et al. 2 Laboratory Diagnosis • • • • • Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes)] Detection of viral genetic material Serologic procedures 3 Microscopy Identification • Light microscopy • Fluorescent microscopy • Electron microscopy 4 Light microscopy • Characteristic CPE • Inclusion Bodies 5 • Cell death Cell rounding Degeneration Aggregation Loss of attachments to substrate • Characteristic histological changes:inclusion bodies in the nucleus or cytoplasm, margination of chromatin • Syncytia: multinucleated giant cells caused by virusinduced cell-cell fusion 6 Fluorescent microscopy • Fluorescent-antibody staining 7 Electron microscopy • Direct detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus. • Immune Electron microscopy: Human rotavirus; HAV; 8 Laboratory Diagnosis • • • • • Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes)] Detection of viral genetic material Serologic procedures 9 Viral isolation and Identification • • • • Viral Growth and Cell culture Viral Detection Viral Identification Interpretation of culture results 10 Systems for the Propagation of Viruses • • • • People Animals: cows, chickens, mice,rats, suckling mice Embryonated eggs Organ and tissue culture Organ culture Primary tissue culture Cell lines: diploid Tumor or ( immortalized ( cell line 11 Viral detection • • • • CPE Hemadsorption Interfere Metabolize of cell 12 TCID50 (Tissue culture infective dose) • TCID50 is defined as that dilution of virus which will cause CPE in 50% of a given batch of cell culture • TCID50= log10 of highest dilution giving 100%CPE +1/2 – (total number of test units showing CPE)/ (number of test units per dilution) 13 Viral identification • • • • • • • • Complement fixation ( Hemagglutination inhibition Neutralization Immunofluorescence ( direct or indirect) Latex agglutination In situ EIA ELISA RIA(radioimmuno 14 Laboratory Diagnosis • • • • • Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes) Detection of viral genetic material Serologic procedures 15 Detection of viral proteins ( antigens and enzymes) • • • • Antigen detection ( ELISA, RIA, Western blot) Hemagglutination and hemadsorption Enzyme activities( reverse transcriptase) Protein patterns( electrophoresis ) 16 Laboratory Diagnosis • • • • • Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes)] Detection of viral genetic material Serologic procedures 17 Detection of viral genetic material • PCR ( Polymerase chain reaction) • RT-PCR (Reverse transcriptase polymerase chain reaction) • Southern ( DNA), Northern(RNA), and dot blots • DNA genome hybridization in situ(cytochemistry) • Electrophoretic mobilities of RNA for segmented RNA viruses( Electrophoresis) • Restriction endonuclease cleavage patterns 18 Laboratory Diagnosis • • • • • Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes)] Detection of viral genetic material Serologic procedures 19 Serologic procedures • If the antibody titer in the convalesent-phase serum sample is at least 4-fold higher than the titer in the acute-phase serum sample, the patient is considered to be infected. • In certain viral diseases, the presence of IgM antibody is used to diagnose current infection • Other nonspecific serologic tests are available 20 Serologic procedures • • • • • • • • Complement fixation ( Hemagglutination inhibition Neutralization Immunofluorescence ( direct or indirect) Latex agglutination In situ EIA ELISA RIA 21 Viruses Diagnosed by Serology • • • • • • Epstein-Barr virus Rubella virus Hepatitis A, B, C, D, and E viruses HIV Human T-cell Leukemia virus Arboviruses ( Encephalitis viruses) 22 Prevention • Successes of the Past • Possibilities for the Future 23 Active immunization Vaccines 24 Overview of Active immunization • Active immunization - administration of antigen resulting in production of a specific immune response with immunologic memory. Response may be cellular or humoral or both. Natural immunity - to diseases you have caught and successfully fought Artificial immunity – Vaccination(vaccines) 25 Attributes of a good vaccine • Ability to elicit the appropriate immune response for the particular pathogen • Long term protection ideally life-long • Safety vaccine itself should not cause disease • Stable retain immunogenicity, despite adverse storage conditions prior to administration • In-expensive 26 LIVE VACCINES • Live attenuated organism • Heterologous vaccines • Live recombinant vaccines • Attributes – live vaccines 27 Live attenuated organism • Organisms whose virulence has been artificially reduced by in vitro Culture under adverse conditions, such as reduced temperature. 28 Heterologous vaccines • Closely related organism of lesser virulence, which shares many antigens with the virulent organism. The vaccine strain replication in the host and induces an immune response that cross reacts with antigens of the virulent organism. • Vaccinia virus /cowpox virus--- Variola virus 29 Live recombinant • Vector 1. bovine vaccine 2. BCG 30 Advantages of Attenuated Vaccines 2-1 •Both cell mediated immunity and antibody response •Activates all phases of immune system. Can get humoral IgG and local IgA •Raises immune response to all protective antigens. Inactivation may alter antigenicity. •More durable immunity; more cross-reactive •Immunity is long lived •Single dose 31 Advantages of Attenuated Vaccines 2-2 • Low cost • Quick immunity in majority of vaccinees • In case of polio and adeno vaccines, easy administration • Easy transport in field • Can lead to elimination of wild type virus from the community 32 Disadvantages of Live Attenuated Vaccine • Mutation; reversion to virulence (often frequent) •Spread to contacts of vaccinee who have not consented to be vaccinated (could also be an advantage in communities where vaccination is not 100%) • Spread vaccine not standardized--may be backmutated • Poor "take" in tropics • Problem in immunodeficiency disease (may spread to these patients) 33 Killed vaccines • The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These vaccines are not infectious and are therefore relatively safe. However, they are usually of lower immunogenicity and multiple doses may be needed to induce immunity. In addition, they are usually expensive to prepare. 34 Killed vaccines • Inactivated organism: rabies virus; epidmic type B encephalitis virus. • Subunit Vaccines: Influenza virus( HA and NA) • Recombinant proteins: HBV 35 Advantages of inactivated vaccines • Gives sufficient humoral immunity if boosters given • No mutation or reversion • Can be used with immuno-deficient patients • These vaccines tend to be able to withstand more adverse storage conditions,Sometimes better in tropics 36 Disadvantages of inactivated vaccines • Many vaccinees do not raise immunity • poor, only antibody, no cell immediated immune response • response is short-lived and multiple doses are needed • No local immunity (important) • Inactivated, therefore can not replicate in the host and cause disease • Failure in inactivation and immunization with virulent virus • Expense: Expensive to prepare 37 New Methods Selection of attenuated virus strain • Varicella • Hepatitis A Use monoclonal antibodies to select for virus with altered surface receptor • Rabies • Reo Use mutagen and grow virus at 32 degrees. Selects for temperature-sensitive virus. Grows in upper respiratory tract but not lower • ‘flu (new vaccine) • respiratory syncytial virus 38 New Methods Passage progressively at cold temperatures TS mutant in internal proteins Can be re-assorted to so that coat is the strain that is this years flu strain 39 PB2 PB1 PA HA NA NP M NS Attenuated Donor Master Strain Attenuated Vaccine Strain: Coat of Virulent strain with Virulence Characteristics of Attenuated Strain X PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS New Virulent Antigenic Variant Strain 40 New Methods Deletion mutants • Suppression unlikely (but caution in HIV) • Viable but growth restrictions Problems • Oncogenicity in some cases (adeno, retro) 41 New Methods • Recombinant DNA •Single gene (subunit) Hepatitis B vaccine S-antigen mRNA cDNA raised in yeast Express plasmid S-antigen mRNA protein 42 Single gene (subunit) - problems • Surface glycoprotein poorly soluble deletion? • Poorly immunogenic • Post-translational modifications • Poor CTL response 43 Single gene (subunit) in expression vector Vaccinate with live virus Canary Pox • Infects human cells but does not replicate • Better presentation • CTL response Vaccinia Attenuated Polio 44 Being developed for anti-HIV vaccine New Methods Chemically synthesized peptide • malaria poorly immunogenic 45 New methods Anti-idiotype vaccine Virus epitope antibody Antibody with epitope binding site 46 Anti-idiotype vaccine cont Antiidiotype antibody antibody Make antibody against antibody idiotype Anti-idiotype antibody mimics the epitope 47 Anti-idiotype antibody cont 2 Use anti-idiotype antibody as injectable vaccine Anti-idiotype antibody Use as vaccine Binds and neutralizes virus Anti-anti-idiotype antibody Anti-anti-idiotype antibody Antibody to anti-idiotype antibody Anti-anti-idiotype antibody 48 New Methods New “Jennerian Vaccines” • Live vaccines derived from animal strains of similar viruses • Naturally attenuated for humans Rotavirus: Monkey Rota 80% effective in some human populations Ineffective in others 49 Due to differences in circulating viral serotypes New Methods New Jennerian Vaccines Bovine parainfluenza Type 3 Bovine virus is: • Infectious to humans • Immunogenic (61% of children get good response) • Poorly transmissable •Phenotypicaly stable 50 New Methods Second Generation Jennerian Vaccines Rotavirus 11 segments of double strand RNA Two encode: • VP4 (hemagglutinin) • VP7 (glycoprotein) Co-infect tissue culture cells Elicit neutralizing antibodies reassortment •10 segments from monkey rotavirus • 1 segment outer capsid protein of each of four major rotavirus strains Efficacy >80% 51 Vaccines • 1796 Jenner: wild type animal-adapted virus • 1800’s Pasteur: Attenuated virus • 1996 DNA vaccines The third vaccine revolution 52 DNA vaccines • DNA vaccines are at present experimental , but hold promise for future therapy since they evoke both humoral and cell-mediated immunity, without the dangers associated with live virus vaccines 53 DNA Vaccines Gene for antigen plasmid Muscle cell Muscle cell expresses protein - antibody made 54 CTL response DNA Vaccines • Plasmids are easily manufactured in large amounts • DNA is very stable • DNA resists temperature extremes so storage and transport are straight forward • DNA sequence can be changed easily in the laboratory. This means that we can respond to changes in the infectious agent • By using the plasmid in the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than purifying that protein and using it as an immunogen. 55 DNA Vaccines • Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so that a broad spectrum vaccine could be produced • The plasmid does not replicate and encodes only the proteins of interest • No protein component so there will be no immune response against the vector itself • Because of the way the antigen is presented, there is a CTL response that may be directed against any antigen in the pathogen. A CTL response also offers protection against diseases caused by certain obligate intracellular pathogens 56 (e.g. Mycobacterium tuberculosis) DNA Vaccines Possible Problems • Potential integration of plasmid into host genome leading to insertional mutagenesis • Induction of autoimmune responses (e.g. pathogenic anti-DNA antibodies) • Induction of immunologic tolerance (e.g. where the expression of the antigen in the host may lead to specific non-responsiveness to that7antigen) 5 DNA Vaccines DNA vaccines produce a situation that reproduces a virallyinfected cell Gives: • Broad based immune response • Long lasting CTL response Advantage of new DNA vaccine for flu: CTL response can be against internal protein In mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins 58 Adjuvants • Certain substances, when administered simultaneously with a specific antigen, will enhance the immune response to that antigen. 59 Adjuvants in common use • Aluminium salts • Liposomes and immunostimulating complexes • Complet Freund’s adjuvant is an emulsion of mycobacteria, oil and water • Incomplete Freund’s adjuvant • Muramyl di-peptide • Cytokines 60 Possible action modes of adjuvant • By trapping antigen in the tissues, thus allowing maximal exposure to dendritic cells and specific T and B lymphocytes • By activating antigen-presenting cells to secrete cytokines that enhance the recruitment of antigen-specific T and B cells to the site of inoculation 61 Smallpox 62 Smallpox • Variolation •1% v. 25% mortality •Life-long immunity • No drift or shift 63 Vaccination Smallpox • Jenner 1796 : Cowpox/Swinepox • 1800’s Compulsory childhood vaccination • 1930’s Last natural UK case • 1940’s last natural US case • 1958 WHO program • October 1977: Last case (Somalia) 64 Smallpox • No animal reservoir • Lifelong immunity • Subclinical cases rare • Infectivity does not precede overt symptoms • One Variola serotype • Effective vaccine • Major commitment by6governments 5 polio • Killed virus vaccine(Salk, 1954) • Live attenuated oral polio vaccine( Sabin, 1957) • The inactivated Salk vaccines is recommended for children who are immunosuppressed. • 66 Polio Vaccine Small RNA virus Some drift…but not too far as non-viable Sabin attenuated vaccine ~ 10 cases vaccine-associated disease per year • 50% vaccinees feces • 50% contacts • Vaccine-associated cases: revertants • 1 in 4,000,000 vaccine infections paralytic polio • 1 in 100 of wt infections Scandinavia: Salk dead vaccine • No gut immunity • 67 Reported cases per 100000 population 100 10 Inactivated (Salk) vaccine Cases per 100,000 population United States Oral vaccine 1 0.1 0.01 0.001 1950 1960 1970 1980 1990 68 Total cases Sweden and Finland 10000 Reported cases Killed (Salk) vaccine 1000 100 10 1 0 1950 1955 1960 1965 1970 1975 69 Reciprocal virus antibody titer 512 Killed (Salk) Vaccine Live (Sabin) Vaccine Serum IgG Serum IgG 128 32 Serum IgM Serum IgM Nasal IgA Serum IgA 8 Serum IgA 2 Duodenal IgA Nasal and duodenal IgA 1 48 Vaccination 96 Days 48 Vaccination 70 96 Sabin Polio Vaccine Attenuation by passage in foreign host More suited to foreign environment and less suited to original host Grows less well in original host Polio: • Monkey kidney cells • Grows in epithelial cells • Does not grow in nerves • No paralysis •Local gut immunity (IgA) Pasteur rabies vaccine also attenuated 71 Salk Polio Vaccine • Formaldehyde-fixed • No reversion 72 Polio Vaccine Why use the Sabin vaccine?: • Local immunity: Vaccine virus just like natural infection • Stopping replication in G.I. Tract stops viral replication TOTALLY • Dead Salk vaccine virus has no effect on gut replication • No problem with selective inactivation • Greater cross reaction as vaccine virus also has antigenic drift • Life-long immunity 73 Measles • Live attenuated virus grown in chick embryo fibroblasts, first introduced in the 1960’s. • Etiology: Measles virus • Incubation: 8 to 12 days • Clinical Manifestations: cough, coryza, conjunctivitis , erythematous maculopapular rash fever ,Koplik Spots ,complictions include Encephalitis, Pneumonia, and SSPE • Treatment: Supportive 74 Mumps • • • • • • • Live attenuated virus developed in the 1960’s MMR vaccine Etiology: Mumps Virus Incubation: 16 to 18 days Clinical Manifestations: swelling of the salivary glands complications include Meningitis, Orchitis, Encephalitis, and Deafness 75 rubella • • • • • Live attenuated virus Etiology: Rubella Virus Incubation: 14 to 21 days Clinical Manifestations: Congenital , cataracts patent ductus arteriosus , deafness mental retardation , Postnatal mild disease , erythematous maculopapular rash , postauricular lymphadenopathy transient polyarthralgias 76 Hepatitis B • Two vaccines are in current use: A serum derived vaccine A recombinant vaccine • Etiology: Hepatitis B Virus • Incubation: 120 days (average) • Clinical Manifestations: jaundice ; anorexia • nausea and vomiting ; malaise • complications include the development of a chronic carrier state with a high risk for Hepatocellular Carcinoma (liver cancer) 77 Hepatitis A • Formalin-inactivated , cell cultured-derived virus, 78 Yellow fever • The 17D strain is a live attenuated vaccine developed in 1937. • It is a highly effective vaccine which is administered to residents in the tropics and travellers to endemic areas. 79 Rabies No safe attenuated strain of rabies virus has yet been developed for human. Vaccines in current use include: a] The neurotissue vaccine b] human diploid cell culturederived vaccine, which is much safer. There are two situation where vaccine is given: a] Post-exposure prophylaxis, followinf the bite of a rabid animal, Hyperimmune rabies globulin may also administered . b] Pro-exposure prophylaxis is used for protection of those occupation puts them at risk of infection with rabies. 80 Influenza • New vaccines are produced every year 81 Varicella-Zoster virus • Not licensed vaccines 82 83 Passive Immunisation 84 Modes of immunization • Passive immunization - administration of antibodycontaining serum to provide immediate, but temporary protection. Doesn't activate a lasting specific immune response. 85 Natural • Provides immunity for diphtheria, tetanus, streptococcus, rubeola (red measles), rubella (German measles), mumps, polio, and others. 86 Artificial • Often used as antitoxins for things such as black widow spider and snake bites, botulism, and tetanus. Important for some infectious diseases such as rabies, since it provides immediate protection rather than waiting the 7-10 days for a protective response to develop from active immunization. 87 Immunoglobulin • “Normal”Immune globulin • Hyper-immune globulin 88 “Normal”Immune globulin Low titres of antibody to a wide range of human viruses • Hepatitis A virus infection • Parvovirus infection • Enterovirus infections (in neonates) • HIV-infected babies 89 Hyper-immune globulin --- high titres of antibody to particular viruses • Zoster immune globulin: prevention of varicella in immunocompromised children and neonates • Human rabies immunoglobulin: post-exposure prophylaxis in an individual who has been bitten by a rabid animal • Hepatitis B immune globulin:non-immune individal who has been exposed to HBV • RSV immune globulin: treatment of respiratory syncitial virus infections in the very young 90 Antiviral Therapy 91 Antiviral Therapy • • • • Antiviral chemotherapy Interferon Gene therapy Chinese Herbs 92 Antiviral chemotherapeutic Agents • Antiviral drugs are available to treat only a few viral diseases. • The reason for this is the fact that viral replication is so intimately associated with the host cell that any drug that interferes significantly with viral replication, is likely to be toxic to the host 93 Targets for chemotherapeutic agents • • • • • Attachment to host cell Uncoating –(amantadine) Synthesis of viral mRNA-(interferon) Translation of mRNA-(interferon) Replication of viral RNA or DNA- (nucleoside anologues) • Maturation of new virus proteins-(protease inhibitors) • Budding , release 94 95 Diseases for which effective therapy is available • AIDS: Zidovudine (((( + Lamivudine (((( + protease inhibitors • • • • • Influenza: Amantadine Herpes simplex virus: Acyclovir Varicella-Zoster virus: Acyclovir Cytomegalovirus : Gancyclovir (((( , Foscarnet ((( Respiratory syncitial virus: Ribavirin (((( 96 Nucleotide analogues • Nucleotide analogues competes with normal nucleotide for incorporation into viral DNA or RNA. 97 Interferon • Direct antiviral effect ( prevents the infection of new cells) by a) degradation of viral mRNA, and b) inhibition of protein synthesis • Enhancement of the specofic immuneresponse by increasing the expression of MHC class I molecules on the surface of infected cells, the interferons increase the opportunity for specifif cytotoxic T cells to recognise and kill infected cells • Chronic hepatitis B and C virus 98 Chinese Herbs • (((((((((((((( • ((( 99 ...
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This note was uploaded on 01/13/2012 for the course MMBIO 361 taught by Professor Bernard during the Fall '11 term at BYU.

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26 - Laboratory Diagnosis 1 Category of Sample • Blood...

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