Lecture 21: Diagnosing Infectious Disease PDF

BIOL371: Microbiology Lecture 21 – Diagnosing infectious disease 1 Topics of today 1. Microbiology and the healthcare environment 2. Immunological and molecular tools for disease diagnosis Materials covered:  Chapter 29.1-29.3, 29.5-29.8  Figures 29.2-29.4, 29.1-29.15, 29.17-29.21, 29.23-29.2...

BIOL371: Microbiology Lecture 21 – Diagnosing infectious disease 1 Topics of today 1. Microbiology and the healthcare environment 2. Immunological and molecular tools for disease diagnosis Materials covered:  Chapter 29.1-29.3, 29.5-29.8  Figures 29.2-29.4, 29.1-29.15, 29.17-29.21, 29.23-29.25  Table 29.1, 29.2 2 Microbiology and the healthcare environment 1. The clinical microbiology laboratory 2. Healthcare-associated infections 3 The clinical microbiology laboratory  Clinical microbiology: a subdiscipline of microbiology focussing on the diagnosis of infectious diseases by identifying pathogenic microbes and advising medical providers on treatment  Standard laboratory practices for handling clinical samples have been established to minimize the risk of accidental laboratory infections  Laboratories are classified according to their containment potential, or biosafety level (BSL), and are designated BSL-1, BSL-2, BSL-3, and BSL-4 4 Working in a Biosafety Level 4 laboratory environment  BSL-4 is the highest level of biological containment; only one facility in Canada (National Microbiology Laboratory, Winnipeg)  Maximum worker protection and pathogen containment  Whole-body sealed suit with an outside air supply and ventilation system  Air locks control all access to the laboratory  All materials leaving the laboratory is autoclaved or chemically decontaminated 5 Microbiology laboratory safety standards Rule Restrict access Implementation Only laboratory workers and trained support personnel have access. Practice good personal hygiene Eating, drinking, applying cosmetics, and manipulating contact lenses are forbidden in the laboratory. Hand washing prevents spread of pathogens. Use personal protective equipment (PPE) Lab coats, gloves, eye protection, and respirators are recommended or required depending on the pathogens being handled. Face masks are part of PPE if respirators are not required. Vaccinate Personnel must be vaccinated against agents to which they may be exposed. Handle specimens safely Assume all clinical specimens are infectious and handle appropriately. After use or exposure, decontaminate specimens, surfaces, and materials by disinfecting, autoclaving, or incinerating. Wash healthcare clothing (scrubs) frequently. Decontaminate 6 Healthcare-associated infections  Nosocomial infection: healthcare-associated infection acquired at a healthcare facility 7 Risk factors for hospital-acquired infections Risk factor for Hospital-acquired infections Rationale Patients Patients are already ill or immunocompromised Newborn infants and the elderly Not fully immune competent Infectious disease patients Pathogen reservoirs Patient proximity Increases cross-infection Healthcare personnel Can transfer pathogens between and among patients; healthcare personnel may be asymptomatic disease carriers Breaching the skin barrier can introduce pathogens Medical procedures (blood draws, etc.) Surgery Anti-inflammatory drug Treatment Antibiotic treatment Exposes internal organs, may introduce pathogens, and causes stress, which lowers resistance to infection Lower resistance to infection May select for resistant and opportunistic pathogens 8 Identification of microbial pathogens  Samples may include blood, urine, feces, sputum, cerebrospinal fluid, or pus  Identification of pathogens require a combination of microbiological immunological, and molecular biological techniques 9 Immunoassays  Immunoassays: use antibodies specific for pathogens or their products for in vitro tests designed to detect specific infectious agents  If an individual is infected, antibodies to that pathogen should become elevated  The antibody titre is a quantitative measure of antibody level and is defined as the highest dilution (lowest concentration) of serum at which an antigenantibody reaction is observed 10 Immunoprecipitation  Precipitation: the interaction of a soluble antibody and soluble antigen to form an insoluble complex  The insoluble complex only occurs when antigen and antibody are in equivalent concentrations The extent of precipitation is a function of antigen and antibody concentration. Inset photo: Precipitation in agarose gel (immunodiffusion). Well S contains antibodies to cells of Proteus mirabilis. Wells A, B, and C contain soluble extracts of P. mirabilis. 11 Agglutination  Agglutination: the visible clumping of a particulate antigen when mixed with antibodies specific for the particulate antigen  Standardized agglutination tests are used to identify blood group antigens, and many pathogens and pathogen products Blood type Percentage of U.S. population Serum Anti A Serum Anti B No aggl O 48 No aggl A 32 Aggl B 16 No aggl AB 4 Aggl utination No aggl utination utination utination Aggl Aggl utination utination utination utination 12 Immunoflorescence  Direct immunofluorescence: the antibody targeted against the surface antigen is covalently linked to the fluorescent dye  Indirect immunofluorescence: the presence of a nonfluorescent primary antibody is detected by a fluorescent secondary antibody directed against the nonfluorescent antibody 13 Applications of immunoflorescence  If the pathogen contains surface antigens reactive with the antibody, the pathogen cells fluoresce  Fluorescent antibodies can be applied directly to infected host tissues, allowing for rapid diagnosis  Fluorescent antibodies can be used in the diagnosis of noninfectious diseases; e.g., malignant cells Cells of Clostridium septicum were stained with antibody conjugated with fluorescein isothiocyanate (yellow-green). Cells of C. chauvoei were stained with antibody conjugated with rhodamine B (red-orange) Detection of cells infected with EpsteinBarr virus using indirect immunofluorescence. The green-stained cells are infected with virus. 14 Enzyme-linked immunosorbent assay  Enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA):  Enzymes covalently bound to antibody molecules to amplify the signals  Very sensitive immunological assay  Widely used in clinical diagnosis and research applications 15 Major versions of enzyme immunoassays A, direct. From a solid phase support, black antibodies are connected to antigens from a patient sample, which are in turn connected to black antibodies. Enzymes are conjugated to the black antibodies, and there is color development due to enzyme activity. B, indirect. Antigens are on a solid phase support. Antibodies from a patient sample are connected to the antigens, and black antibodies are connected to the patient sample antibodies. There are enzymes conjugated to the black antibodies, and color development due to enzyme activity. C, sandwich. Antigens are on a solid phase support, connected to antibodies from a patient sample, which are further connected to another antigen. Enzymes are conjugated to the antigens and there is color development due to enzyme activity. 16 Rapid tests  Rapid tests: similar to enzyme immunoassays  Results in minutes instead of hours  Generally not as specific or sensitive  Reagents are absorbed to support materials  Body fluid is applied to the support matrix  Matrix contain soluble antibody (or antigen depending on the method) A patient specimen containing a mixture of antigens is applied to conjugated to a coloured molecule the sample well of a support matrix. Capillary action pulls the liquid sample through the matrix, and specific antigen (if present) binds (chromophore) soluble, chromophore-labeled antibodies. The labeled antigen– antibody complexes diffuse through the matrix and bind a line of  Matrix also contains a line of fixed fixed antibodies. A colored line becomes visible as the antibody which the antigen-antibody concentration of labeled complex builds, indicating a positive test for the antigen. Unbound labeled antibodies bind a second line of complexes bind to, detect colour fixed antibody as a control 17 change Monoclonal antibodies  An antigen has many epitopes and stimulate the production of many antibodies (polyclonal antibodies), some of which may cross-react with other antigens  Monoclonal antibody: by isolating single clones of B cells that are fused with cancer cells to make immortalized cell lines that produce a single type of antibody  The hybridoma generated can be cultured, passes through animal as a tumour, stored frozen for later use  Monoclonal antibodies are increasingly being used in clinical diagnostics for immunological typing of bacterial pathogens and as therapeutics 18 Nucleic acid hybridization  Hybridization methods identify specific pathogens in patient samples by using unique nucleic acid probes to detect pathogen DNA Membrane filter assay. The reporter can be a radioisotope, a fluorescent dye, or an enzyme Dipstick assay. The capture probe contains a poly(A) tail that hybridizes to a poly(T) oligonucleotide affixed to the dipstick. Binding of the target DNA–reporter complex is usually detected as a visible color change. Quantitative and reverse transcription PCR  Presence of amplified gene segment confirms presence of pathogen  Reverse transcriptase PCR (RT-PCR) uses pathogen-specific RNA to make c D N A  Quantitative real-time PCR (qPCR) uses fluorescently labeled PCR products and gene-specific primers for amplification results in hours