Microbiology Lecture Slides PDF

General Principles of Laboratory Dx WHY DO WE CARE? GENERAL PRINCIPLES OF LABORATORY DIAGNOSTICS  CLASS OBJECTIVES  Describe the four diagnostic principles  Assess the performance of different diagnostic tests  Aka apply sensitivity, specificity, PPV, NPV (with math, but I’ll try to pi...

General Principles of Laboratory Dx WHY DO WE CARE? GENERAL PRINCIPLES OF LABORATORY DIAGNOSTICS  CLASS OBJECTIVES  Describe the four diagnostic principles  Assess the performance of different diagnostic tests  Aka apply sensitivity, specificity, PPV, NPV (with math, but I’ll try to pick easy numbers so it’s not difficult math)  Understand when to use tests from each of the 4 diagnostic principles (especially clinically) & the meaning of the results  Be able to diagnose infections by culture (phenotypically)  Describe molecular and genetic approaches to studying bacteria ASSESSING THE PERFORMANCE OF DIAGNOSTIC TESTS  True positive- pathogen positive, test positive  True negative- pathogen negative, test negative  False positive- pathogen negative, test positive (Type 1 Error)  False negative- pathogen positive, test negative (Type 2 Error)  Sensitivity- ability of test to correctly identify those with disease (true positive rate)  Specificity- ability of test to correctly identify those without the disease  Positive predictive value- probability that subjects with a positive test will truly have the disease  Negative predictive value- probability that subjects with a negative test truly don’t have the disease DIAGNOSTIC SENSITIVITY AND SPECIFICITY: HOW RELIABLE IS THE TEST?  Interpretation of test results requires an understanding of the test’s reliability prior to diagnosis or treatment  No test is perfect; every testing method produces several false-positive & false-negative results  How much emphasis should be placed on a particular test depends on sensitivity & specificity  Sensitivity- probability that the test will be positive in a patient who has the disease in question  Specificity- probability that the result will be negative in a patient who does not have the disease  Sensitivity & specificity of a test can be determined by using a 2 × 2 table (a, b, c, & d are actual numbers of observations, not proportions) TRUE VS FALSE  A true positive is an outcome where the model correctly predicts the positive outcome  A true negative is an outcome where the model correctly predicts the negative outcome  A false positive is an outcome where the model incorrectly predicts the positive outcome  A false negative is an outcome where the model incorrectly predicts the negative outcome  LET’S APPLY IT!  Imagine the evaluation of a diagnostic test developed to predict the dreaded disease Examinus paralysis, commonly known as “brain freeze,” among students about to take an exam. Data from previous experience indicates that: True positive True negative -------------------------- -------------------------- True pos + false neg False pos + true neg WHAT IS THE SENSITIVITY? WHAT IS THE SPECIFICITY THE FOUR DIAGNOSTIC PRINCIPLES 1. Microscopic 2. Cultivation and 3. Measurement of a 4. Detection of pathogen- examination of patient identification of pathogen-specific immune specific macromolecules samples. microorganisms from response in the patient. in patient samples patient samples.  1. DIAGNOSING INFECTIONS BY MICROSCOPY- STAINS  Gram stains & acid-fast stains  Presence of bacteria in a normally sterile body fluid (i.e. CSF, blood, urine)  Less useful from a nonsterile body site (ie skin)  Staining properties & morphology help with species identification & empirical selection of antibiotics  A diagnosis  Giemsa stain- systemic protozoal infections  Lugol’s iodine stains- intestinal helminths  Silver stains- systemic fungal infection  1. DIAGNOSING INFECTIONS BY MICROSCOPY- ANTIBODY BASED IDENTIFICATION  Specific antibodies enhance accuracy of microscopic identification  A monoclonal antibody to epitope= most specific  A polyclonal antibody can be least specific  Depends on what you’re looking for and cross-reactivity  Direct immunofluorescence: fluorophore is conjugated to the antibody  Indirect immunofluorescence: unlabeled primary antibody is added, then an anti-primary fluorophore secondary antibody is added to bind to the unlabeled primary 1. DIAGNOSING INFECTIONS BY MICROSCOPY- ANTIBODY BASED IDENTIFICATION. DIRECT & INDIRECT IMMUNOFLUORESCENCE Immunofluorescence pic because I love it (Yes, I took this one, no, it’s not bacteria)  2. DIAGNOSING INFECTIONS BY CULTURE  Agar-based media and in a broth medium under both aerobic and anaerobic conditions  Blood culture  Direct inoculation of blood into nutrient broth & incubation to check for microbial growth  Sub-cultured & transferred to agar plates for identification  OR lysis-centrifugation technique- RBCs lysed and remaining dense material inoculated in agar medium & plated  Culture identification  Phenotypic properties: motility, utilization of various nutrient substrates, enzymes produced, etc.  Antibody-based techniques  Selective media can identify specific cultures 2. COMMON CULTURES: EMB & MACCONKEY AGAR 2. DIAGNOSING INFECTIONS BY CULTURE  Antimicrobial sensitivity testing- tested for susceptibility to antimicrobial agents 3. MEASURING THE ANTIBODY RESPONSE TO INFECTION- WESTERN BLOT  Serology- diagnostic examination of blood serum, esp. regarding immune response to pathogens  Western blot- one of the most specific serologic methods  Pathogen’s antigens are separated based on size using electrophoresis  Antigens are then “blotted” onto solid support & incubated with patient’s serum to see if antibodies bind to pathogen-specific or cross- reactive antigens 3. MEASURING THE ANTIBODY RESPONSE TO INFECTION- ELISA  Enzyme-linked immunosorbent assay (ELISA)  Solid-phase assay= pathogen or pathogen antigen’s fixed to solid support  Patient’s serum incubated. Antibodies for pathogen will bind to antigen, those that don’t will be washed away  Enzyme-labeled anti-antibodies bind to patient antibodies  When enzyme binds catalyzes production of visibly colored compounds  Measure the amount of bound enzyme based on color  Second antibody may be made specific for IgG or IgM or other immunoglobulins TEST YOUR KNOWLEDGE #1  You are a Family Medicine Physician.Your healthy, 30-year-old, biologically male patient presents to your office complaining of severe gastrointestinal pain, nausea, vomiting, and diarrhea. After talking to your patient about signs & symptoms, if he knows anyone currently sick with norovirus, and things he ate in the last 24-48 hours, you begin to become suspicious that the bagged salad your patient ate the night before might be the culprit as there is a salad recall currently in effect. If you were doing a stool culture, which agar would you use to support your theory that he has an E.coli infection? (and why, so we can discuss in class) A. Bile Esculin Agar B. EMB agar C. Nutrient broth agar D. MacConkey agar  4. DIAGNOSING INFECTION BY DETECTING PATHOGEN MACROMOLECULES (& GENETIC TESTING) A. Antigen detection tests B. Nucleic Acid-Based Diagnosis of Infection C. Microarrays D. Next-Gen Sequencing  A. ANTIGEN DETECTION TESTS  Like reverse serologic tests  Specific antibodies used to capture antigen from a patient’s sample  Ex. Simple agglutination assay  Negative- no antigen binds to antibodies  Positive- antigen binds causing clumping  Prozone- too much antigen than antibody, no clumping occurs, false negative  To fix: dilute & it becomes positive Latex agglutination test- uses antibody-coated latex beads to detect capsular material  A. ANTIGEN DETECTION TESTS- ELISA & EIA  Enzyme-linked immunosorbent assay (ELISA)- quantify antigen in solution  Enzyme immunoassays (EIA)- used to visualize and quantify antigens  Patient sample co-incubated with enzyme labeled antigen. Will compete with binding to antibody on solid support  No antigen= no decrease in color  Antigen in patient’s sample= decrease in color TEST YOUR KNOWLEDGE #2  You perform antimicrobial sensitivity testing on a patient’s sample. These are the results. Which antibiotic(s) would be the most effective to prescribe the patient? (Just give me the letter(s)) B. NUCLEIC ACID-BASED DIAGNOSIS OF INFECTION  DNA is two strands. When you heat it, the strands separate  When the temperature lowers (annealing), they reconnect “hybridization” (they HAVE to be complementary to each other)  Using a DNA probe (short single-stranded DNA sequence) you can hybridize it to a “target” sequence  DNA probe test was first nucleic acid-based test used  In situ hybridization can be done in tissue  C. NUCLEIC ACID AMPLIFICATION- PREMISE BEHIND PCR  Sometimes you don’t have enough of the specific nucleic acid in a sample to use direct detection with DNA probe to so need to amplify uses the concepts previously discussed Amplified sequences = amplicons  POLYMERASE CHAIN REACTION (PCR)  Real-time PCR (aka quantitative PCR aka qPCR) (NOT “RT- PCR” which is reverse-transcriptase PCR)  Targeted for specific segment of DNA bound by two primers  Commercially available assays available to detect different bacteria & viruses in patient samples (time saving)  Can use fluorescent-labeled probe  Combines steps of amplification & amplicon analysis  Helps quantify the target nucleic acid sequence by measuring # of cycles required to reach threshold of fluorescent detection  Fun fact: a “bad” PCR was used for reasonable doubt in OJ Simpson case TO PCR OR NOT TO PCR… NUCLEIC ACID BASED DIAGNOSIS OF INFECTION  PCR DEBATE  In med school, the basic scientists (PhDs) will tell you to perform PCRs, but the clinicians will tell you to treat  This is nuanced & depends on context  E.g., if a patient is in shock & dying of a bacterial infection, you don’t have time to wait for a PCR, you would treat with broad-spectrum  However, if a patient is presenting with a new viral infection and you need to know what it is, or if a patient is not responding to treatment of a disease PCR would be the correct answer choice to determine what it is  In an ideal world, you could start broad spectrum treatment, while sending off PCR & switch patient to more specific treatment once results are in. Boards questions don’t always allow that option, so read carefully for the context! PROS & CONS OF PCR VS. SEROLOGY VS. ANTIGEN C. MICROARRAYS  Since bacteria have conserved sequences, you have the potential to amplify products from any bacterial species  You could then hybridize to the microarray containing sequences from hundreds of bacterial species  Can help you determine which one (or ones) is (are) present in patient sample  D. NEXT-GEN SEQUENCING  Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation TEST YOUR KNOWLEDGE #3  Which of these latex agglutination tests are positive or negative?  ALSO A SCIENTIST Jane Hinton, DVM (1919-2003) -DVM from University of Pennsylvania  One of the first two African-American women to earn a DVM  Granddaughter of slaves  Her father Dr. William Augustus Hinton opened the first “Medical Laboratory Techniques” courses open to women (“not a woman’s job”)  Co-developed Mueller-Hinton agar used to isolate Neisseria bacteria.  Mueller-Hinton agar is the gold standard for antibiotic testing