Chapter 17 Procedures for Identifying Pathogens and Diagnosing Infection 1 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
17.1 An Overview of Clinical Microbiology Methods of identifying unknown microbes fall into three categories: 1. Phenotypic observable microscopic and macroscopic characteristics 2. Genotypic genetic make up 3. Immunological serology; antibody-antigen reactions 2
1. Phenotypic Methods Microscopic morphology fresh or stained microorganisms from specimen; shape, size, stain reaction, 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 3
2. Genotypic Methods Assess genetic make-up PCR, use specific primers for virulence genes Culture is not necessary Precise, automated methods, quick results 4
3. Immunological Methods Specific antibodies are used to detect antigens Easier than testing for the microbe itself 5
On the Track of the Infectious Agent: Specimen Collection Sampling body sites or fluids for suspected infectious agent Results of lab tests depend on specimen collection, handling, transport, and storage HANDLE WITH CARE!! Aseptic procedures should be used Figure 17.1 Specimen Collection Saliva Sputum Nasopharynx Throat (tonsils) Skin: Swab Tamper-evident seal Plastic case a. Sampling sites b. Sterile transport swab Clean catch Insert Fig 17.1 Catheter Blood Vaginal swab or stick Spinal tap (Cerebrospinal fluid) Feces Long swab with rayon tip Transport medium Squeeze container to release medium Skin: Scalped (b) 6 (a)
Specimen Collection 7
Figure 17.2 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: most likely Confirmatory: know exactly 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 reaction to microbe Clinical signs and symptoms 8
17.2 Phenotypic Methods Immediate direct examination of Specimen Microscopic: differential and special stains, example: Gram, Spore, direct antigen testing Sample from lesion or exudate Figure 17.3 Direct fluorescent antibody test Flourescent monoclonal Ab solution specific for syphilis spirochete Wash Positive fluorescence Negative No fluorescence (a) Syphilis spirochete Not syphilis spirochete (b) Fred Marsik/Visuals Unlimited 9
Phenotypic Methods Cultivation of Specimen Colony appearance, growth requirements, appropriate media Biochemical testing Physiological reactions to nutrients as evidence of the absence or presence of enzymes 10
Figure 17.4 Rapid Tests and Identification (a) (b) Gram (+) Analytab Products, a division of Sherwood Medical Rods Gram ( ) Figure 17.5 Flowchart to separate gram-positive and gram-negative bacteria 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 Bacillus Clostridium Mycobacterium Nocardia Lactobacillus Listeria Pseudomonas Alcaligenes Acinetobacter Corynebacterium Propionibacterium Escherichia Enterobacter Citrobacter Proteus Salmonella Erwinia Shigella Klebsiella Vibrio Campylobacter 11
Phenotypic Methods Miscellaneous tests Antimicrobial sensitivity: Kirby-Bauer method, measure zone of inhibition in mm (millimeter), determine R, I, S. 12
17.3 Genotypic Methods DNA analysis PCR DNA and RNA analysis Ribosomal RNA Comparison of the sequence of nitrogen bases in ribosomal RNA, determine organism Figure 17.6 PCR testing to identify pathogens 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 20 Dr.Anwar Huq and Bradd J. Haley 13
17.4 Immunological Methods Serology in vitro diagnostic testing of serum Antibodies have extreme specificity for antigens Visible reactions 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 14
Figure 17.8 Specificity and sensitivity in immune testing Abs Ag1 Ag2 Ab for Ag2 Ags (a) (b) a. Specificity between antigen and antibody antigen present b. Ab can detect ag in dilute amounts - titer 15
Figure 17.7 Serological Testing Patient s serum antibody content unknown Prepared Known antigen Isolated colony, identity unknown Antibodies of known identity Ag ANTISERUM Macroscopic reaction Macroscopic reaction Ag Molecular reaction Molecular reaction (a) In serological diagnosis of disease, a blood sample is scanned for the presence of antibody using an antigen of known specificity. A positive reaction is usually evident as some visible sign, such as color change or clumping, that indicates a specific interaction between antibody and antigen. (The reaction at the molecular level is rarely observed.) (b) Ab Ag on microbe An unknown microbe is mixed with serum containing antibodies of known specificity, a procedure known as serotyping. Microscopically or macroscopically observable reactions indicate a correct match between antibody and antigen and permit identification of the microbe. 16
General Features of Immune Testing Agglutination testing antibody cross links whole-cell antigens, forming complexes that settle out and form visible clumps Blood typing (ABO blood system), some bacterial and viral diseases Agglutination* Whole cell Epitope + The Tube Agglutination Test Reaction + + + + + + + Unagglutinated cells Antigen Antibody Microscopic appearance of clumps Precipitation* Cell-free molecule in solution Epitope Antigen + Antibody Agglutinated cells Dilution (b) 1/20 1/40 1/80 1/160 1/320 1/640 Control (no serum) The tube agglutination test. A sample of patient s serum is serially diluted with saline. 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 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 control, which will be cloudy and clump-free. The titer is equivalent to the denominator of the dilution of the last tube in the series that shows agglutination. Microscopic appearance of precipitate (a) Agglutination involves clumping of whole cells; precipitation is the formation of antigen-antibody complexes in cell free solution. Both reactions can be observed by noticeable clumps or precipitates in test tubes (see (b) and figure 17.10a). *Although IgG is shown as the Ab, IgM is also involved in these reactions. Figure 17.9 Cellular / molecular view of antigen-antibody complexes 17
17.5 Immunoassays: Tests of Great Sensitivity Extremely sensitive to detect trace antigens and antibodies Enzyme-linked immunosorbent assay (ELISA) enzyme-antibody complex produces a colored product when an enzyme-substrate reaction occurs Indirect Capture 18
ELISA Methods Figure 17.15 Methods of ELISA testing A (a) Indirect ELISA, comparing a positive vs. negative reaction. This is the basis for HIV screening tests. Known antigen is adsorbed to well. Serum samples with unknown antibodies Sample A Well A Well B Sample B (b) Microtiter ELISA Plate with 96 Tests for HIV Antibodies. Colored wells indicate a positive reaction. B Hank Mogan/Science Source/Photo Researchers, Inc. Well is rinsed to remove unbound (nonreactive) antibodies. (c) Capture or Antibody Sandwich ELISA Method. Note that an antigen is trapped between two antibodies. This test is used to detect the measles virus. 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 substrate for enzyme is added. Enzymes linked to indicator Ab hydrolyze the substrate, which releases a dye. Wells that develop color are positive for the antibody; colorless wells are negative. (1) ( 2) Enzyme Enzyme-linked antibody specific for test antigen then binds to different antigenic site forming a sandwich. Enzyme s substrate ( ) is added, and reaction produces a visible color change ( ) 19
In vivo Testing Antigens are introduced directly into the body to determine the presence or absence of antibodies Tuberculin skin test, allergy testing look for skin reaction, such as redness and/or swelling at the injection site. 20
17.6 Viruses as a Special Diagnostic Case Figure 17.17 Methods used to diagnose viral infections (1) Cells infected with herpesvirus (2) Cells infected wit herpesvirus # 6 Viruses present special difficulties because they are not cells Viruses are labor intensive to culture in the laboratory (g) ELISA method (a) Signs and symptoms: Patient is observed for manifestations of typical virus infections, such as mumps and herpes simplex (above). Cell culture Embryo (f) Culture techniques: Viruses require a living host to multiply. Dr. Hermann/CDC Zaki Salahuddin. Laboratory of TTumor Cell Biology/National Cancer Institute (b) Cells taken from patient are examined for evidence of viral infection, such as inclusion bodies (1) or virus antigen detected by fluorescent staining (2). Rotavirus Bryon Skinner/CDC Dane particle Hepatitis B CDC 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. Rolan Davis and Mike Moore/Rabies Laboratory,Kansas Stste University (e) A reverse transcriptase PCR fingerprint showing positive rabies test results for 10 patient samples. Genelabs Diagnostics Pvt Ltd. Western blot for HIV (see figure 17.12) (d) Serological testing for antibodies. 21