Chapter 15-Diagnosing Infections PDF

Chapter 15-Diagnosing Infections 15.1 Methods of Microbial Identification 1. Phenotypic Methods Observation of traits that an organism is expressing in appearance and behavior Examples: ○ Types of enzymatic activities it can carry out...

Chapter 15-Diagnosing Infections 15.1 Methods of Microbial Identification 1. Phenotypic Methods Observation of traits that an organism is expressing in appearance and behavior Examples: ○ Types of enzymatic activities it can carry out ○ Physical conditions it thrives in ○ Antibiotics it is susceptible to ○ Chemical composition of its walls and/or membranes 2. Immunologic Methods (think of immune system) A patient sample is tested for the presence of specific antibodies to a suspected pathogen Often easier than testing for the microbe itself Laboratory kits are available for immediate identification of a number of pathogens 3. Genotypic Methods Examining genetic material Advantages of genomic methods over phenotypic methods: ○ Growing the microorganism is not always necessary ○ Genotypic methods are increasingly automated, producing rapid results that are often more precise than phenotypic methods Viable non culturable (VNC): microbes that cannot be grown in the laboratory that are identified by genotypic methods 15.2 First Steps: Specimen Collection Success of identification and treatment depends on how specimens are collected, handled, stored, and cultured: ○ Aseptic(free of microbes) techniques like keeping containers and tools sterile is important Only the infected site should be sampled, and not the surrounding areas Sampling sites: Salvia Sputum Swab Blood Urine Catheter Skin swab Spinal tab Feces Vaginal swab Skin scalpel Punch biopsy Phenotypic Methods: Immediate Direct Examination of Specimen Direct microscopic observation is one of the most rapid methods of determining characteristics Stains most often used: ○ Gram stain ○ Acid-fast stain Selective and differential growth: ○ Specialized media: used to grow a pathogen present in small numbers ○ Selective media: encourage the growth of only the suspected pathogen ○ Differential media: used to visually distinguish microbes Dichotomous Key ○ Flowchart to separate primary genera of various cocci: Combines results of gram staining, growth on differential media, and simple enzymatic tests  Biochemical Testing ○ Physiological reactions of bacteria to nutrients and other substrates: Provide evidence of enzyme systems present in a species Phenotypic Test ○ Mycobacterial growth indicator tube (MGIT): detects the growth of the slow-growing mycobacterium tuberculosis ○ Antimicrobial Susceptibility Testing: useful in determining which drugs will be used in treatment ○ Phage Typing: uses bacteriophages to detect and identify bacteria Immunologic Methods: Serology Involves in vitro(in a lab) testing of serum, urine, cerebrospinal fluid, whole tissues, and salvia for the presence of specific antibodies The presence of antibodies can reveal the history of a patient’s contact with microorganisms or other antigens Immunochromatography ○ Lateral flow test found in drugstore pregnancy tests and rapid COVID test ○ If the sample contains the correct antigen, it will bind to the antibodies and continue on to the next “station” in the cartridge ○ Next stage contains a third molecule that is impregnated on the paper in a stripe pattern Antibody Titers ○ Concentration of antibodies in a sample ○ Determined by diluting patient serum into test tubes or wells of a microtiter plate containing equal amounts of bacterial cells (antigen) ○ The more a serum sample can be diluted and still react with antigen, the greater the concentration of antibodies ○ Used to diagnose autoimmune disorders and determine past exposure to certain diseases Serotyping ○ Antigen-antibody technique for identifying, classifying, and subgrouping certain bacteria into categories called serotypes ○ Widely used in identifying Salmonella species and strains Western Blot Test  ○ Use of electrophoresis to detect the presence antigens or antibodies ○ Used to detect HIV antibodies Enzyme-Linked Immunosorbent Assay (ELISA) ○ Uses an enzyme-linked indicator antibody to visualize antigen-antibody reactions ○ Relies on a solid support such as a microtiter plate that can adsorb the reactants General Features of Immune Testing ○ The most effective diagnostic tests have a high degree of specificity and sensitivity: Specificity: property of a test to focus only on a certain antibody or antigen, and not react with an unrelated or distantly related antigen Sensitivity: detection of even minute quantities of antibodies or antigens in a specimen; reflects the degree to which a test will detect every positive person Genotypic Methods: Polymerase Chain Reaction (PCR) PCR results in the production of numerous identical copies of DNA or RNA molecules within hours Hybridization: Probing for Identity ○ Hybridization: makes it possible to identify a microbe by analyzing segments of its genetic material (FISH) ○ Probes: small segments of DNA or RNA known to be complementary to the specific sequences of the nucleic acid isolated from a microbe