Diagnosis of Bacterial Infections PDF 2024

VPRP096 Introduction to Microbiology: Diagnosis of Bacterial Infections Dr. Ricardo Gutiérrez, Microbl., MSc, Ph.D. Assistant Professor [email protected] Spring semester, 2024 Diagnosis of bacterial infections nf ormation Rapid Accurate Is it an infectious disease? Which pathogen is? What is...

VPRP096 Introduction to Microbiology: Diagnosis of Bacterial Infections Dr. Ricardo Gutiérrez, Microbl., MSc, Ph.D. Assistant Professor [email protected] Spring semester, 2024 Diagnosis of bacterial infections nf ormation Rapid Accurate Is it an infectious disease? Which pathogen is? What is the antimicrobial sensitivity profile? Does it have any antibiotic resistance? Which treatment plan should I recommend? Any prevention measurements? How about control plan? Is it a zoonosis? Is it notifiable disease? Regular submission of clinical specimens for isolation, identification and susceptibility testing will improve and update this knowledge Diagnosis of bacterial infections Pre-analytical Analytical Post-analytical phase phase phase Animal species Clinical characteristics Disease state Epidemiology Biospecimen type Anatomical site Selection of methodology Sample collection Selection of methodology Processing Preparation Analysis Result & review Reporting Interpretation Diagnosis Treatment Diagnosis of bacterial infections SAMPLE COLLECTION Choice for specimen collection depends on Clinical symptoms Typeof pathogen Location of infection Duration of infection Diagnostic tests to be performed Common specimens for bacterial infections: skin, ear swab, urine, wound swabs, blood, milk Proper specimen collection is the FIRST and KEY step for accurate diagnostic testing! Diagnosis of bacterial infections SAMPLE COLLECTION Tissues Scraps/Swabs/Impression Blood Specimen Types Vomitus/sputum Feces Transudate/Exudates Urine/Misc. fluids Diagnosis of bacterial infections SELECTION OF METHODOLOGY Detection of the agent 1. Direct detection of the bacteria 2. Sample cultivation 3. Taxonomic identification of pathogen ✓ ✓ ✓ ✓ ✓ Infection type? Test availability? Sensitivity? Specificity? Time and costs? Detection of host immune response 1. Serology: detection of humoral immunity 2. Detection of cell-mediated immunity Diagnosis of bacterial infections Examination of stained or unstained preparations (‘smears’) Detection of the agent 1. Direct detection of the bacteria Microscopy and differential staining Fluorescent antibody staining Single most important and cost-effective procedure Gives an idea of: Bacterial density (estimation of bacterial numbers) Bacterial morphology Host immune response (cellular reaction) Common staining: Gram-staining Ziehl-Neelsen staining (acid-fast staining) Fluorescent antibody staining Electron microscopy (generally used only for research) Diagnosis of bacterial infections DIRECT MICROSCOPY OBSERVATION Milk sample from a bitch. Gram-staining. Big red arrow indicates the presence of Gram-negative bacilli. Small red arrow indicates an immune system cell (foamy cell) Mycobacterium sp. revealed by fluorescent microscopy in a sputum sample. Using fluorescence, the sensitivity to detect the bacteria is improved, and it is easy to differentiate them from other artifacts or cells. Diagnosis of bacterial infections SAMPLE CULTIVATION Many pathogens can be isolated from clinical samples in the laboratory, using simple media. This process is known as cultivation or isolation of bacteria. Media can be liquid (broth medium) or solid (agar medium). To successfully isolate bacteria, we must provide the bacteria their optimal nutrients, atmosphere and temperature. Bacteria grow faster on liquid media. Nevertheless, in solid medium, bacteria can be distinguished by phenotypic characteristics. Diagnosis of bacterial infections SAMPLE CULTIVATION Bacterial colony Colony Forming Unit (CFU) = 1 bacterium 1 bacterium 1 CFU Diagnosis of bacterial infections BACTERIAL COLONY: Diagnosis of bacterial infections TYPES OF MICROBIAL SOLID GROWTH MEDIA General- purpose media Allows growth of a large number of non-fastidious bacteria Selective media Contains inhibitory compounds for certain groups. Any medium can become selective (just add an inhibitory substance) Differential media Allow the distinguishment of different bacterial groups Diagnosis of bacterial infections Energy source: Lactose or Peptones Inhibitory compounds: Bile salts and crystal violet, which largely inhibit the growth of the Gram-positive bacteria. Differentiation: pH indicator (if lactose is consumed pH will be acidic; if peptones are consumed, pH will be basic) EXAMPLE: MacConkey Agar (Selective & Differential medium) Media can be both selective and differential! Non-lactose fermenting bacteria (colorless colony) Lactose fermenting bacteria (pink color colony) Diagnosis of bacterial infections EXAMPLE: Blood agar is a general purpose, enriched medium often used to grow fastidious organisms and to differentiate bacteria based on their hemolytic properties Some general proposed medium may give extra information for differentiate the bacteria Diagnosis of bacterial infections Incubation: Most pathogens growth at 37°C (mesophiles) Incubation time range from 18h-48h Agar medium Routine samples are cultured on three different solid media: 1. General-purpose or enriched medium 2. Differential and selective medium for Gram-negative 3. Differential and/or selective medium for Gram-positive 2nd Incubation Primary culture: Mixed culture, with pathogen and microbiota 2nd culture: May have the pure pathogenic bacteria Diagnosis of bacterial infections CULTURE OF FASTIDIOUS ORGANISM: Chocolate agar Anaerobic chamber to work and incubate “strict anaerobic bacteria” Atmospheric jar. Gas converter is added to change the composition of the atmosphere. Chopped-meat broth Incubator with controlled CO2 % Diagnosis of bacterial infections IDENTIFICATION Biochemical characterization 2nd culture: May have “pure” pathogenic bacteria Proteomic characterization (MALDI-TOF) PCR Fatty-acid characterization (GC) Real-time PCR Diagnosis of bacterial infections IDENTIFICATION Fluorescent antibody staining 2nd culture: May have “pure” pathogenic bacteria Enzyme-linked immunosorbent assay (ELISA) Agglutination tests Monoclonal antibodies for antigen-recognition for detection and/or identification Goals: SEROTYPING & Antigen detection Diagnosis of bacterial infections All bacteria in nature Culturable (~1%) Unculturable (~99%) The term ‘unculturable’ is used to describe bacteria that are not grown on artificial media till date Diagnosis of bacterial infections SELECTION OF METHODOLOGY Detection of host immune response 1. Serology: detection of humoral immunity 2. Detection of cell-mediated immunity Let´s do a immune response recap… Diagnosis of bacterial infections BRIEF RECAP ON IMMUNE SYSTEM PATHWAYS Usually, viruses Exposure! (time 0) Diagnosis of bacterial infections BRIEF RECAP ON IMMUNE SYSTEM PATHWAYS Example! Air-borne pathogen LYMPH NODE LYMPH NODE https://www.youtube.com/watch?v=xM0bozu8Q6Q&t=633s Diagnosis of bacterial infections BRIEF RECAP ON IMMUNE SYSTEM PATHWAYS https://www.youtube.com/watch?v=xM0bozu8Q6Q&t=633s Diagnosis of bacterial infections BRIEF RECAP ON IMMUNE SYSTEM PATHWAYS https://www.youtube.com/watch?v=xM0bozu8Q6Q&t=633s Diagnosis of bacterial infections SELECTION OF METHODOLOGY SEROLOGY Diagnostic identification of antibodies in serum Detection of host immune response 1. Serology: detection of humoral immunity 2. Detection of cell-mediated immunity Evidence of exposure to an infectious agent does not necessarily confirm an etiological role for that agent in the animal sampled. Collection of 2 serum samples with 2 weeks apart and a fourfold rise of antibody titer are indicative for recent exposure to an infectious agent (SEROCONVERSION) Diagnosis of bacterial infections Sample 2 Sample 3 Sample 1 Immunoglobulin (Ig) levels Exposure! (time 0) IgG IgM Time Seroconversion No seroconversion Diagnosis of bacterial infections SEROLOGY METHODS Diagnosis of bacterial infections SEROLOGY METHODS Antibody titer & Seroconversion Antibody titer: measurement of antibody level (quantity). Expressed as the reciprocal of the highest dilution of serum that positively reacts in a specific test. The higher the antibody response, the higher the titer e.g. a titer of 1280 means that the serum sample remains positive when diluted 1:1280 Seroconversion: a four-fold rise of antibody titer indicates antibody development after exposure to a pathogen or antigen Diagnosis of bacterial infections SELECTION OF METHODOLOGY DETECTION OF CELL-MEDIATED IMMUNITY Detection of host immune response ‘Cell-mediated immunity’ refers to the adaptive immune response that is independent of antibodies but involves immune cells that specifically recognize, target, and clear infected host cells. 1. Serology: detection of humoral immunity 2. Detection of cell-mediated immunity Intracellular infectious agents such as certain bacteria (e.g., Listeria monocytogenes or Mycobacterium tuberculosis) and viruses require a cell-mediated immunity response for their elimination. Diagnosis of bacterial infections CELL-MEDIATED INMUNITY BASED METHODS Delayed-type hypersensitivity https://www.youtube.com/watch?v=mzC9NgY2Rj0 Diagnosis of bacterial infections CELL-MEDIATED INMUNITY BASED METHODS Delayed-type hypersensitivity https://www.youtube.com/watch?v=mzC9NgY2Rj0 Diagnosis of bacterial infections CELL-MEDIATED INMUNITY BASED METHODS https://www.youtube.com/watch?v=mzC9NgY2Rj0 Inflammation (swelling) Diagnosis of bacterial infections TUBERCULIN SKIN TEST Testing for bovine tuberculosis (Mycobacterium bovis) Measure of cell-mediated immunity (delayed hypersensitivity) Obligatory in the EU Procedure Day 1: two sites are clipped, and tuberculin injected (M. bovis and M. avium antigens) Day 4: measure skin reaction ➔ if bovine site > 4 mm = animal is reactor Diagnosis of bacterial infections TUBERCULIN SKIN TEST PRINCIPLE https://www.youtube.com/watch?v=QgES9AwWB8s Diagnosis of bacterial infections INTERFERON GAMMA TEST (IFN-ϒ) Whole blood test that can aid in diagnosis of Mycobacterium infections Based on the amount of IFN-ϒ that is released or on the number of cells releasing IFN-ϒ One visit: results within 24h