Transcript - MediPIET
Public Health Microbiology Identification Techniques Introduction to Intervention Epidemiology Tunis, 3 November 2014 Prof. Olfa Bahri Aziza Othmana Hospital - Tunis [email protected] INTRODUCTION PROBLEMATIC – The same symptomatology for different infectious diseases – Different symptoms for the same infectious disease – Different course to follow according to pathogen involved LABORATORY DIAGNOSTIC+++ INTRODUCTION ROLE OF LABORATORY DIAGNOSTIC+++ − Identification of pathogens responsible for the disease − Characterization of pathogens (important especially in case of outbreaks) − Surveillance of the evolution of infectious disease − Detection of new pathogens AIM OF THIS PRESENTATION To decribe and understand The different identification methods of viruses, bacteria and parasites The principles of the most common identification methods The advantages and limitations of the methods In which situation typing is relevant IDENTIFICATION AND CHARACTERISATION • First part Identification Diagnostic Second part Characterisation Typing Laboratories Laboratories Identify the microorganism • responsible for suspected infection • Obtain an isolate of the pathogen (pure culture) define the strain to which the isolate is belonging to • phenotypic and/or genotypic characterisation DIRECT versus INDIRECT Methods DIRECT methods detect …. • whole pathogen • particles of the agent (cell wall components) • genetic components of the agent (DNA) the pathogen DIRECT versus INDIRECT Methods DIRECT methods detect …. INDIRECT methods detect … • particles of the agent • components of the agent (DNA) • antibodies • cell mediated immune response the pathogen a response induced by exposure to the pathogen DIRECT IDENTIFICATION METHODS 1. Direct observation of the agent 2. Propagation of the agent (culture) 3. Detection of nucleic acids (DNA / RNA) 4. Detection of antigen (agent particles) WHERE DO WE LOOK ? SUITABLE SPECIMEN COLLECTION 1. DIRECT OBSERVATION OF THE AGENT TOOLBOX • Macroscopic evaluation • Light microscopy • Electron microscopy 1. DIRECT OBSERVATION OF THE AGENT MACROSCOPIC EVALUATION Appearance of sample (consistence, sludge or sediment, odour, …) Rice water stools Vibrio cholerae infection? Abnormal urine Bacterial urinary infection? Abnormal CSF Bacterial meningitis? 1. DIRECT OBSERVATION OF THE AGENT LIGHT MICROSCOPY • No direct observation of viruses or phages • Bacterial detection – Without coloration: Mobility, Form of the bacteria – Coloration methods • Classical coloration (Methylen blue, Gram stain+++) • Specific coloration (Parasites, Mycobacterium…) • Usually no species identification 1. DIRECT OBSERVATION OF THE AGENT LIGHT MICROSCOPY • Differential staining Acid-fast Mycobacterium tuberculosis (1000x) Giemsa (parasites) Plasmodium falciparum 1. DIRECT OBSERVATION OF THE AGENT LIGHT MICROSCOPY • Differential staining Gram staining Gram + Staphylococcus Gram E. coli 1. DIRECT OBSERVATION OF THE AGENT LIGHT MICROSCOPY Cell wall of gram negative bacteria Cell wall of gram positive bacteria LIGHT MICROSCOPY 1. DIRECT OBSERVATION OF THE AGENT LIGHT MICROSCOPY •Advantages • Rapid (minutes to one hour) • Inexpensive •Disadvantages • Low sensitivity (minimum 105 bacteria/ml necessary) • Usually no species identification • No direct observation of viruses or phages 1. DIRECT OBSERVATION OF THE AGENT ELECTRON MICROSCOPY Examination of viruses (referral laboratories) Rotavirus Norovirus Ebolavirus 1. DIRECT OBSERVATION OF THE AGENT ELECTRON MICROSCOPY • Advantages • Broad • Can detect viruses • Reference test for novel infections • Disadvantages • EM not usual in laboratories • Specific and expensive equipment • Low sensitivity (minimum 106 virus/ml necessary) Example: Identification of SARS-Cov - 2002 – 2003: Outbreak of SARS throughout the world - Unknown pathogen?? Electron microscopy: Coronavirus 1. DIRECT OBSERVATION OF THE AGENT PROPAGATION OF THE AGENT • Using of different support allowing a culture of microorganisms: – For viruses (Cell cultures, embryonated eggs) – For bacteria (liquid or solid media) • Importance of conditions for growth (type of support used, temperature, nutrition, atmosphere…..) 1. DIRECT OBSERVATION OF THE AGENT PROPAGATION OF THE AGENT embryonated eggs 1. DIRECT OBSERVATION OF THE AGENT PROPAGATION OF THE AGENT Cell being cultured in Petri dish 1. DIRECT OBSERVATION OF THE AGENT PROPAGATION OF THE AGENT ECP for poliovirus ECP for CMV 2. PROPAGATION OF THE AGENT Culturing bacteria in vitro on solid or in liquid media Broth Agar plate HOW WE START? PRIMARY CULTURE ISOLATE Pure culture Genus & Species e.g. Salmonella enterica Diagnostic/Identification Clinical microbiology laboratory Reference laboratory HOW DO WE SORT? PRIMARY CULTURE ISOLATE STRAIN Strain A Strain B Genus & species & ssp & serotype Pure culture A: Salmonella enterica ssp. enetrica ST Typhimurium Genus & Species B: Salmonella enterica ssp. enetrica ST Enteritidis e.g. Salmonella enterica Diagnostic/Identification Clinical microbiology laboratory Reference laboratory Typing/Characterisation Reference laboratory 2. PROPAGATION OF THE AGENT Growth on solid media - colony structure and shape changes Colony shape Colony- edge shape B. subtilis growth morpohology on Yeast-extract agar 1% Shape -˃ aspect of diferentiation Growth -˃ specific colony morphology (B. subtilis) Surface shape 2. PROPAGATION OF THE AGENT PHENOTYPIC IDENTIFICATION Further identification after isolation of the agent • Identification tests for both: G+ and G- anaerobic bacteria, yeast and fungi based on : • Enzymatic reactions (catalase, coagulase,…) • Metabolism of certain compounds (glucose, lactose,…) In test tubes or prefabricated cards (Api) 2. PROPAGATION OF THE AGENT PHENOTYPIC IDENTIFICATION Advantages • Broad (most common causes of infections) • Semi-quantitative • Inexpensive Disadvantages • Long turn-around if slow growth rate (e.g. M.tuberculosis) • Can not be used for unculturable agents • Appropriate biosafety containment facilities necessary 3. DETECTION OF NUCLEIC ACIDS TOOLBOX • PCR • Real-time PCR • Sequencing 3. DETECTION OF NUCLEIC ACIDS NUCLEIC ACID AMPLIFICATION (PCR) Amplification of DNA by Polymerase Chain Reaction (PCR) PCR 3. DETECTION OF NUCLEIC ACIDS NUCLEIC ACID AMPLIFICATION Detection of amplified specific DNA/RNA by gel electrophoresis and size estimation Multiplex PCR Single PCR M 1. 2. 3. 4. 1 2 3 4 Leptospira interrogans sample A Leptospira interrogans sample B Positive control - 330 bp band Leptospira interrogans sample C M 1. 2. 3. 4. 5. 1 2 3 4 5 Positive control Listeria monocytogenes, Neisseria meningitidis Haemophilus influenzae type b Streptococcus pneumoniae 3. DETECTION OF NUCLEIC ACIDS REVERSE HYBRIDIZATION 3. DETECTION OF NUCLEIC ACIDS NUCLEIC ACID AMPLIFICATION Advantages • Sensitive • Rapid (conventional PCR 1,5 - 4h) • Small volume patient material needed • Detection, quantification and genetic characterisation of pathogen Disadvantages • High risk of contamination • Expensive 3. DETECTION OF NUCLEIC ACIDS REAL TIME PCR - Avantadges: - Specificity - Sensitivity - Detection and quantification of pathogens - Less risk of contamination 3. DETECTION OF NUCLEIC ACIDS GENETIC CHARACTERISATION OF PATHOGEN Sequencing 3. DETECTION OF NUCLEIC ACIDS GENETIC CHARACTERISATION OF PATHOGEN 3. DETECTION OF NUCLEIC ACIDS GENETIC CHARACTERISATION OF PATHOGEN 4. DETECTION OF ANTIGENS TOOLBOX • Agglutination • ELISA • Immunofluorescence HOST RESPONSE INDIRECT DETECTION 4. DETECTION OF ANTIGENS AGGLUTINATION Direct and latex agglutination (slides, cards) Vibrio cholerae Staphylococcus aureus 4. DETECTION OF ANTIGENS ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 4. DETECTION OF ANTIGENS ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Detection of bacterial, viral or parasite antigen / toxin Coated antibody Antigens contained in patients sample immunoglobulin coupled to an enzyme 4. DETECTION OF ANTIGENS IMMUNOFLUORESCENCE ASSAY (IFA) Detection of antigens of viruses (HSV) in infected cells Herpesvirus 4. DETECTION OF ANTIGENS IMMUNOFLUORESCENCE ASSAY (IFA) Detection of antigens specific of rabies in infected cells 4. DETECTION OF ANTIGENS Advantages • Rapid (minutes) • Easy to perform/low training needs • Can be performed at patient site Disadvantages • Sensitivity can be low • Decreased specificity due to cross-reactions • Pitfalls in interpretation of result missed by non-trained personnel INDIRECT IDENTIFICATION METHODS 1. Detection of antibodies 1. DETECTION OF ANTIBODIES TOOLBOX • ELISA • Complement fixation • Haemagglutination inhibition • Neutralization 1. DETECTION OF ANTIBODIES ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Advantages: - Détection of different types of antibodies (IgG, IgM, IgA….) - Titration of antibodies 5. ANTIBODY RESPONSE COMPLEMENT FIXATION http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter31/animation_quiz_4.html Antibody dilution Fixation: test pos No fixation: test neg THANK YOU Prof. Olfa Bahri Aziza Othmana Hospital - Tunis [email protected]