Phenotypic Methods. Genotypic Methods. Immunological Methods 10/10/2013. Clinical Microbiology. Specimen Collection

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1 0/0/0 Clinical Microbiology Chapter 7 Procedures for Identifying Pathogens and Diagnosing Infection Methods of identifying unknown microbes fall into three categories:. Phenotypic observable microscopic and macroscopic characteristics. Genotypic genetic make up. Immunological serology; antibody-antigen s Microscopic morphology fresh or stained microorganisms from specimen; shape, size, stain, cell structures Macroscopic morphology colony appearance; texture, size, shape, pigment, growth requirements Physiological/biochemical characteristics detection of presence or absence of particular enzymes or metabolic pathways Chemical analysis analyze specific chemical composition; cell wall peptides, cell membrane lipids Genotypic Methods Assess genetic make-up Culture is not necessary Precise, automated methods, quick results Immunological Methods Specific antibodies are used to detect antigens Easier than testing for the microbe itself Specimen Collection Sampling body sites or fluids for suspected infectious agent Results depend on specimen collection, handling, transport, and storage Aseptic procedures should be used Saliva Sputum Nasopharynx Throat (tonsils) Skin: Swab Tamper-evident seal Plastic case Blood Insert Fig 7. Spinal tap (Cerebrospinal fluid) Feces Vaginal swab or stick Long swab with rayon tip Clean catch Transport medium Catheter Squeeze container to release medium Skin: Scalped

2 0/0/0 Specimen Collection Overview of Laboratory Techniques Routes taken in specimen analysis Direct tests (microscopic, immunologic, or genetic) Cultivation, isolation, and identification (general and specific tests) Two categories of results Presumptive Confirmatory Direct Testing Microscopic Gram stain Acid-fast stain Fluorescent Ab stain Gene probes Macroscopic Direct antigen DNA typing Immunologic and serological tests (antibody titer) are performed on blood and other fluids Specimen Patient Culture/Isolation Tests on isolates Biochemical Serotyping (Slide) Antimicrobic sensitivity PCR analysis Phage typing Animal inoculation In vivo test for to microbe Clinical signs and symptoms 7 8 Concept Check: If you examine whether or not your organism has a particular DNA sequence in order to identify it, you are using techniques. Immediate direct examination Microscopic differential and special stains Gram, DFA, direct antigen testing Sample from lesion or exudate A. Direct Flourescent monoclonal Ab solution specific for syphilis spirochete B. Genotypic C. Immunological Positive fluorescence Wash Negative No fluorescence D. Phenotypic 9 Syphilis spirochete Not syphilis spirochete Fred Marsik/Visuals Unlimited 0 Cultivation of Specimen Colony appearance, growth requirements, appropriate media Biochemical testing Physiological s to nutrients as evidence of the absence or presence of enzymes Rapid Tests and Identification Analytab Products, a division of Sherwood Medical Rods Gram () Gram ( ) Sporeformer Non-sporeformer Aerobic, oxidase () Facultative anaerobic, oxidase ( ) (ferment glucose) Aerobic, Oxidase () ferment glucose curviform shapes Acid-fast Not acid-fast Motile Nonmotile Regular Pleomorphic Pseudomonas Alcaligenes Acinetobacter Bacillus Mycobacterium Lactobacillus Corynebacterium Clostridium Nocardia Listeria Propionibacterium Escherichia Enterobacter Citrobacter Proteus Salmonella Erwinia Shigella Klebsiella Vibrio Campylobacter

3 ANTISERUM 0/0/0 Genotypic Methods Miscellaneous tests Phage typing Animal inoculation Antimicrobial sensitivity DNA analysis Hybridization Probes complementary to the specific sequences of a particular microbe PCR DNA and RNA analysis Ribosomal RNA Comparison of the sequence of Dr.Anwar Huq and Bradd J. Haley nitrogen bases in ribosomal RNA Immunological Methods Serology in vitro diagnostic testing of serum Antibodies have extreme specificity for antigens Visible s include precipitates, color changes, or the release of radioactivity Tests can be used to identify and to determine the amount of antibody in serum titer Ags Abs Ag Ag Ab for Ag Patient s serum antibody content unknown Serological Testing Prepared Isolated colony, Known identity unknown antigen Antibodies of known identity Immune Testing Agglutination testing antibody cross links whole-cell antigens, forming complexes that settle out and form visible clumps Blood typing, some bacterial and viral diseases Ag Agglutination* Whole cell Epitope The Tube Agglutination Test Reaction Unagglutinated cells Macroscopic Macroscopic Antigen Antibody Molecular Molecular Ag Ag on microbe Ab In serological diagnosis of disease, a blood sample is scanned for the presence of antibody using an antigen of known specificity. A positive is usually evident as some visible sign, such as color change or clumping, that indicates a specific interaction between antibody An unknown microbe is mixed with serum containing antibodies and antigen. (The at the molecular level is rarely observed.) of known specificity, a procedure known as serotyping. Microscopically or macroscopically observable s indicate a correct match between antibody and antigen and permit 7 identification of the microbe. Agglutinated Microscopic appearance of clumps cells Dilution /0 /0 /80 /0 /0 /0 Control (no serum) Precipitation* The tube agglutination test. A sample of patient s serum is serially diluted with saline. Cell-free molecule in solution The dilution is made in a way that halves the number of antibodies in each subsequent tube. An equal amount of the antigen (here, blue bacterial cells) is added Epitope to each tube. The control tube has antigen, but no serum. After incubation and centrifugation,each tube is examined for agglutination clumps as compared with the Antigen control, which will be cloudy and clump-free. The titer is equivalent to the denominator Antibody of the dilution of the last tube in the series that shows agglutination. Microscopic appearance of precipitate Agglutination involves clumping of whole cells; precipitation is the formation of antigen-antibody complexes in cell free solution. Both s can be observed by noticeable clumps or precipitates in test tubes (see and figure 7.0a). *Although IgG is shown as the Ab, IgM is also involved in these s. 8

4 Gillies and Dodd's Bacteriology illustrated, th edition, figure, Elsevier National Institute Slide Bank/The Welcome Centre for Medical Sciences 0/0/0 7 Immune Testing Precipitation tests soluble antigen is made insoluble by an antibody VDRL Most precipitation s are done in agar gels I. I. In one method of setting up a double-diffusion test, wells are punctured in soft agar, and antibodies (Ab) and antigens (Ag) are added in a pattern. As the contents of the wells diffuse toward each other, a number of s can result, depending on whether antibodies meet and precipitate antigens. Concept Check: In agglutination s, the antigen is a ; in precipitation s, it is a. II. Precipitation bands III. Side view Test Control Ab Ag Test C II. Example of test pattern and results. Antigen (Ag) is placed in the center well and antibody (Ab) samples are placed in outer wells. The control contains known Abs to the test Ag. Note bands that form where Ab/Ag meet. The other wells (, ) contain unknown test sera. One is positive and the other is negative. Double bands indicate more than one antigen and antibody that can react. III. Actual test results for detecting infection with the fungal pathogen Histoplasma. Numbers and are controls and,,, and are patient test sera. Can you determine which patients have the infection and which do not? A. Soluble molecule, whole cell B. Whole Cell, soluble molecule C. Bacterium, virus D. Protein, carbohydrate 9 0 Step Immunoelectrophoresis () Direction of electrophoresis ( ) Step sample separated next to trough Antiserum added to trough sample The Western Blot for Detecting Proteins Electrophoretic separation of proteins, followed by an immunoassay to detect these proteins Highly specific and sensitive Sites of specific antibody binding will appear as a pattern of bands Second test used to verify HIV status gp0 gp0 p p p gp p9 p p p7 Control HIV- Specific band SRC Days Albumin Alpha globulin (a globulin) lgm Gamma globulin (g globulin)(lgg) Genelabs Diagnostics Pte Ltd. Successive tests on an HIV patient over 0 days reveals an increase in band intensity over time. This is due to continued formation of anti-hiv antibodies. Complement Fixation Stage Reaction System Stage Lysin mediated hemolysis Steps of test. Test antigen reacts with test antibody. Contents of tube from (.) are mixed with sheep s Complement used up in first stage, no hemolysis Unfixed complement, hemolysis Sheep red blood cells Positive patient s serum with lysins on surface Ab Ag Complement Lysins (unrelated to Ab in stage ) Ab/Ag Complement complex fixed to Ab Negative patient's serum Complement fixes to antibodies; s do not lyse No hemolysis () Antibody Complement fixes to s; hemolysis occurs No Ab A g Complement Lysins No Ab/Ag complex Hemolysis Free complement ( ) No antibody is fixed by lysins No fixation present on s

5 Dr. Hermann/CDC Rolan Davis and Mike Moore/Rabies Laboratory,Kansas Stste University B. Huard, T. McKee, C. Bosshard, S. Durual, T. Matthes, S. Myit, O. Donze, C. Frossard, C. Chizzolini, C. Favre, R. Zubler, J. P. Guyot, P. Schneider and E. Roosnek, APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa, J Clin Invest. 008;8(8): Copyright 008, American Society for Clinical Investigation Zaki Salahuddin. Laboratory of TTumor Cell Biology/National Cancer Institute Bryon Skinner/CDC Genelabs Diagnostics Pvt Ltd. 0/0/0 CDC Fluorescent Antibody and Immunofluorescent Testing Fluorescent antibody Monoclonal antibody labeled by a fluorescent dye FABs can be used for direct or indirect testing Visible fluorescence Immunoassays Extremely sensitive to detect trace antigens and antibodies Radioimmunoassay (RIA) antigens or antibodies labeled with radioactive isotopes Unknown antigen (usually cell or tissue) Direct Testing Ab in No Known Ag Ab in serum Indirect Testing Fluorescent microscopy Antibody labeled with fluorescent dye Ab fluorescently labeled; specific for Ab Positive Negative Abattaches to Abvisible fluorescence Ab cannot attachesno fluorescence CHEMICON International, Inc. (c) Indirect Immunofluorescence of specimen Enzyme-linked immunosorbent assay (ELISA) enzyme-antibody complex produces a colored product when an enzyme-substrate occurs Indirect Capture ELISA Methods Indirect ELISA, comparing a positive vs. negative Microtiter ELISA Plate with 9 Tests for HIV Antibodies.. This is the basis for HIV screening tests. Colored wells indicate a positive. Well Well A B Known antigen is adsorbed to well. Sample Sample A B samples with unknown A antibodies B Hank Mogan/Science Source/Photo Researchers, Inc. Well is rinsed (c) Capture or Antibody Sandwich ELISA Method. to remove unbound Note that an antigen is trapped between two antibodies. (nonreactive) This test is used to detect the measles virus. antibodies. Antibody specific to test antigen is adsorbed to well. Indicator antibody linked to enzyme attaches to any bound antibody. Test antigen is added; if complementary, antigen binds to antibody. Wells are rinsed to remove unbound indicator antibody. A colorless Enzyme substrate for enzyme is added. Enzyme-linked antibody specific for test antigen then binds to different antigenic site forming a sandwich. Enzymes linked to indicator Ab hydrolyze the substrate, which releases a dye. Enzyme s substrate ( ) is Wells that develop added, and produces color are positive () ( ) a visible color change ( ) for the antibody; colorless wells are negative. 7 Tests that Differentiate T Cells and B Cells Rosette formation Mix T cells with sheep red blood cells Fluorescent techniques Differentiates T and B cells and subgroups them Rosette T cell Phototake s 8 In vivo Testing Virus Diagnosis () Cells infected with () Cells infected wit herpesvirus herpesvirus # Antigens are introduced directly into the body to determine the presence or absence of antibodies Tuberculin skin test, allergy testing Viruses present special difficulties because they are not cells Signs and symptoms: Patient is observed for manifestations of typical virus infections, such as mumps and herpes simplex (above). (g) ELISA method Cells taken from patient are examined for evidence of viral infection, such as inclusion bodies () or virus antigen detected by fluorescent staining (). Rotavirus Hepatitis B Viruses are labor intensive to culture in the laboratory Embryo Cell culture (f) Culture techniques: Viruses require a living host to multiply. Dane particle Filament (c) Electron microscope is used to view virus directly. Viruses are sufficiently unique in structure that they can be differentiated to family or genus. 9 (e) A reverse transcriptase PCR fingerprint showing positive rabies test results for 0 patient samples. Western blot for HIV (see figure 7.) (d) Serological testing for antibodies. 0

6 0/0/0 Concept Check: The Western blot test can be used to identify A. Unknown antibodies B. Unknown antigens C. Specific DNA D. Both A. and B.