Diagnosis of Bacterial Infections PDF

VPRP096 Introduction to Microbiology: Diagnosis of Bacterial Infections Dr. Ricardo Gutiérrez, Microbl., MSc, Ph.D. Assistant Professor [email protected] Diagnosis of bacterial infections nf ormation Is...

VPRP096 Introduction to Microbiology: Diagnosis of Bacterial Infections Dr. Ricardo Gutiérrez, Microbl., MSc, Ph.D. Assistant Professor [email protected] Diagnosis of bacterial infections nf ormation Is it an infectious disease? Rapid Which pathogen is? What is the antimicrobial sensitivity profile? Accurate 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 Selection of methodology Reporting Clinical characteristics Processing Interpretation Disease state Preparation Diagnosis Epidemiology Analysis Treatment Biospecimen type Result & review Anatomical site Selection of methodology Sample collection 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 SELECTION OF METHODOLOGY Detection of the agent Detection of host immune response 1. Direct detection of the bacteria 1. Serology: detection of humoral immunity 2. Sample cultivation 2. Detection of cell-mediated immunity 3. Taxonomic identification of pathogen Diagnosis of bacterial infections Detection of the agent Examination of stained or unstained preparations (‘smears’) 1. Direct detection of the bacteria Single most important and cost-effective procedure Microscopy and differential staining Gives an idea of: Fluorescent antibody staining Bacterial density (estimation of bacterial numbers) Bacterial morphology Host immune response (cellular reaction) Diagnosis of bacterial infections Common staining: DIRECT MICROSCOPY OBSERVATION Gram-staining Ziehl-Neelsen staining (acid-fast staining) Fluorescent staining Milk sample from a bitch. Mycobacterium sp. revealed by fluorescent microscopy in a sputum sample. Gram-staining. Big red arrow indicates the Using fluorescence, the sensitivity to detect the bacteria is improved, and it is presence of Gram-negative bacilli. Small red arrow easy to differentiate them from other artifacts or cells. indicates an immune system cell (foamy cell) 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 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 Proteomic characterization Fatty-acid characterization (GC) 2nd culture: (MALDI-TOF) May have “pure” pathogenic bacteria PCR Real-time PCR Diagnosis of bacterial infections IDENTIFICATION Fluorescent antibody staining Enzyme-linked immunosorbent assay (ELISA) 2nd culture: May have “pure” pathogenic bacteria Agglutination tests Monoclonal antibodies for antigen-recognition for detection and/or identification Goals: SEROTYPING & Antigen detection Diagnosis of bacterial infections Culturable (~1%) All bacteria in nature 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 SELECTION OF METHODOLOGY SEROLOGY Diagnostic identification of antibodies in serum Detection of host immune response Evidence of exposure to an infectious agent does not necessarily confirm an etiological role for that agent in the animal sampled. 1. Serology: detection of humoral immunity Collection of 2 serum samples with 2 weeks apart and a four- 2. Detection of cell-mediated immunity fold rise of antibody titer are indicative for recent exposure to an infectious agent (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 Sample B Sample C Sample A IgG Immunoglobulin (Ig) levels Exposure! (time 0) IgM Time Seroconversion No seroconversion Diagnosis of bacterial infections SELECTION OF METHODOLOGY DETECTION OF CELL-MEDIATED IMMUNITY ‘Cell-mediated immunity’ refers to the adaptive immune response that is independent of antibodies but involves immune cells that Detection of host immune response specifically recognize, target, and clear infected host cells. Intracellular infectious agents such as certain bacteria (e.g., Listeria 1. Serology: detection of humoral immunity monocytogenes or Mycobacterium tuberculosis) and viruses require a cell-mediated immunity response for their elimination. 2. Detection of cell-mediated immunity Diagnosis of bacterial infections TUBERCULIN SKIN TEST PRINCIPLE https://www.youtube.com/watch?v=QgES9AwWB8s 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 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