[MICRO] Principles of Diagnostic Microbiology.pdf

MICROBIOLOGY 08/20/2024. MOD 1: PRINCIPLES...

MICROBIOLOGY 08/20/2024. MOD 1: PRINCIPLES OF DIAGNOSTIC MICROBIOLOGY Ma. Teresa A. Barzaga, M.D., FPSP Trans Group/s: Volunteers 1 I. DIAGNOSTIC MICROBIOLOGY B. GUIDELINES IN SELECTION AND COLLECTION OF Concerned with the etiologic diagnosis of an infection CLINICAL SPECIMENS Encompasses the characterization of thousands of 1. IDENTIFICATION OF SPECIMEN agents that cause, or are associated with infection ○ Techniques used to identify these agents vary from Type of specimen (exact source and nature of laboratory to laboratory and depend on the clinical specimen) symptoms of the patient Collection date and time Good communication is important between laboratory Laboratory number and clinician. Laboratory findings ○ The physician must communicate with the Tests requested laboratory on his diagnosis so the laboratory can Ordering physician use the proper tools to help in treatment. 2. COLLECTION, HANDLING, AND STORAGE OF SPECIMENS A. FUNCTIONS OF A MICROBIOLOGY LABORATORY Diagnostic medical microbiology: a discipline that The physician must know when and how to collect identifies etiologic agents of diseases. specimens, what tests to request, and how to interpret lab results. FUNCTIONS OF A MICROBIOLOGY LABORATORY GUIDELINES IN COLLECTION, HANDLING, AND 1 Etiologic diagnosis Isolation and STORAGE OF SPECIMENS of an infectious identification of disease infectious agent Most important step: proper Demonstration of collection of specimen immunologic response Collected from active site of infection that is most likely to 2 Set guidelines for rational use of antimicrobials yield the organism Specimens are selected based on signs and symptoms, and 3 To test specimens from patients for should be representative of the microorganisms that are/or may be a cause of the disease process illness 1 Collection ○ If symptoms point to a certain organ, collect from 4 To provide information whenever appropriate about that organ the in-vitro activity of antimicrobial drugs against Timing (acute phase of the microorganisms identified illness) ○ BEFORE administration of 1. WHO CAN REQUEST LABORATORY SERVICES? antimicrobial agents Those who can request laboratory services include: Amount ○ All licensed physicians, dentists, and Proper tools and containers optometrists ○ All public health nurses and physicians Viability may be lost if specimen assistants Delivery, is delayed ○ Local Health Departments Transport, Transport media stabilize ○ Communicable Disease Specialists Processing conditions and prevent drying 2 Patient details The more rapidly a specimen ○ Hospital no. (Immediately is plated onto appropriate ○ First name and family name within 2 hrs) media, the better the chance ○ Sex of isolating the pathogen ○ Date of birth/Age ○ Address Appropriate conditions ○ For females: whether pregnant or lactating All specimens can be kept at ○ Details of illness room temperature EXCEPT ○ Presenting signs/symptoms 3 Storage urine, stool, viral specimens, ○ Duration/date of onset sputum, swabs, foreign devices, ○ Recent travel gastric biopsies, and rectal swabs Microbiology - Mod 1 Principles of Diagnostic Microbiology 1 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited. 3. INSTRUCTIONS FOR COLLECTION OF SPECIMEN Contains pathogen but is contaminated with nonpathogenic INSTRUCTIONS FOR COLLECTION flora 1 Safety considerations 3 Samples Primary site of infection: in an from normal area known to be colonized with 2 Selection of appropriate anatomic site flora sites many organisms (pharynx and large intestines) 3 Type of swab or transport medium Infections may be caused by: ○ Bacteria 4 Transport instructions including time and ○ Viruses temperature ○ Fungi ○ Parasites Pathogens: 5 Labeling instructions ○ Exogenous: from environmental, animal 6 Special instructions such as patient preparation sources, or other persons ○ Endogenous: from the Quality of the specimen is crucial. normal flora The results from a laboratory are only as good as the specimen it receives. Therefore, the quality of Pathogen and nonpathogenic flora submitted specimens should always be taken into are mixed at the site of infection consideration. D. SAMPLING SITES AND NORMAL FLORA 3.1 LABELING Upon receiving the specimen and requisition with SAMPLING SITES AND NORMAL FLORA complete data, record it in the microbiology log book in numerical order. Normally Blood and bone marrow The number assigned to the specimen is written on the sterile sites Peritoneal and pulmonary cavity specimen container and the requisition form, culture Middle and Inner ear media containers, and culture media plates. Urinary bladder Date and time of processing and the name of the Accessory nasal sinuses patient should be written clearly on all culture plates, Trachea, bronchi, alveoli tubes, slides, or whatever used in the processing of Cerebrospinal fluid (CSF) specimens. Kidneys Prostate C. CATEGORIES OF SPECIMEN Uterus Larynx Posterior Urethra SPECIMENS Sites with Mouth and Nose 1 Direct tissue Collected from: normal External ear canal or fluid ○ normally sterile tissues commensal Female genital tract samples (lungs, liver) flora External genitalia ○ body fluids (CSF, blood) Eye Methods: Throat and oropharynx ○ Needle aspiration Skin ○ Surgical biopsy Urethra Gastrointestinal tract (GIT) Contains only the pathogen (+) findings: diagnostic E. METHODS FOR CIRCUMVENTING INDIGENOUS (-) findings: exclude infection at the FLORA suspected site CIRCUMVENTING INDIGENOUS FLORA 2 Indirect Pathogen is localized in sterile Samples areas but must pass through a site containing normal flora in 1 Antisepsis 1-2 minutes contact time with order to be collected a disinfectant (70% alcohol Degree of contamination is or betadine) related to the skill with which the normal flora site was bypassed 2 By-pass Transtracheal aspiration in specimen collection. procedures Open biopsy Bypassing normal flora (invasive) for areas Percutaneous needle requires extra effort, and with normal flora biopsy results require interpretive Suprapubic aspiration evaluation of contamination. of urine Microbiology - Mod 1 Principles of Diagnostic Microbiology 2 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.  3 Culture specifically Group A streptococci in 2 Saliva Saliva is unacceptable for for organisms throat culture. Submit deep cough or known to be induced sputum. etiologic agents 3 Multiple urine, Multiple urine, stool, sputum, 4 Quantitate culture E.g. quantitative urine stool, sputum, or or routine throat specimens results cultures routine throat sent on the same day from the specimens same source from the same 5 Decontamination 4-10% NaOH or hypochlorite patient. for mycobacterial solution to kill the bacteria in cultures the sputum samples and only OTHER SPECIMENS UNSATISFACTORY FOR allowing TB organisms to grow CULTURES 6 Selective media SS agar, TCBS 1 Specimens such Specimens in fixative Salmonella Shigella as: (formalin) Agar: for growth of only Dried out swabs salmonella or shigella Foley catheter tips TCBS 24 hour urine/sputum for (Thiosulfate-Citrate-Bile routine bacteria, or fungi Salts-Sucrose): for Urine held 2 hours or growth of Vibrio cholerae more at room temperature Fluids received in F. VIRAL DIAGNOSIS culturette tubes Easier — essentially little normal viral to confuse Swab material for interpretation of results anaerobic culture not in Lack of normal viral flora simplifies interpretation the proper anaerobic Cultures of viruses are easier than cultures of bacteria transport Gram stains for Neisseria II. CRITERIA FOR REJECTION OF SPECIMEN gonorrhoeae on vaginal In general, specimens for the microbiology laboratory or anal crypt specimens are unacceptable if any of the following conditions apply: are not diagnostic and will not be performed Stool specimens for GENERAL CRITERIA FOR REJECTION OF SPECIMEN culture from a patient who has been an inpatient 1 Patient Erroneous entry greater than 5 days information on Incomplete data Anaerobic cultures on requisition slip vaginal, cervical, urine *unless suprapubic tap), 2 Specimen Improperly collected sputum, or fecal specimen Dried up/dried swab Placed in fixative Pooled specimen (24 III. FLOW OF PROCEDURES IN LABORATORY hour) DIAGNOSIS OF INFECTIOUS DISEASES Insufficient quantity A. DIRECT EXAMINATION 3 Container Improper container Direct examination frequently provides the most rapid Leaking container indication of microbial infection. Quality of the specimen can be assessed. 4 Transport Improper temperature Microbiologists and clinicians can be given an early Inappropriate transport indication of what may be wrong with the patient. containers used Work-up of specimens can be guided by comparing Delay between collection what grows in culture to what was seen on smear. and arrival in the The field of microbiology has been largely defined by the laboratory development and use of the microscope. ○ The examination of specimens by microscopic 5 Processing Produce information of methods rapidly provides useful diagnostic questionable medical value information. In order for bacteria to be seen, they must be stained. ○ Staining techniques permit microorganisms to be A. OTHER CRITERIA FOR REJECTION OF SPECIMEN seen more clearly, because without staining bacteria are difficult to see at the magnifications BACTERIOLOGY used for their detection. ○ Although simple one-step stains can be used, using 1 Unsuitable blood Blood received in blood culture differential stains, e.g. gram stain, is more culture bottles bottles unsuitable for fungal common. isolation ○ Cellular morphology and gram stain characteristics are often used to categorize Microbiology - Mod 1 Principles of Diagnostic Microbiology 3 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.  stained organisms into groups (e.g. streptococci, 1.2 ACID FAST STAIN staphylococci, and clostridia). Used to stain acid-fast bacteria whose cell wall contain 1. DIFFERENTIAL STAINING METHODS mycolic acids which render the cells resistant to decolorization even with acid alcohol decolorizers 1.1 GRAM STAINING Identifies organisms that retain carbol-fuschin dye after acid or organic solvent disruption Differentiates between: Weak initial staining and strong retention is related to ○ Gram (+) bacteria: organisms with thick the high lipid content of the mycobacterial cell wall. peptidoglycan cell walls with numerous teichoic acid Modifications of this procedure allow the differentiation cross linkages that offer resistance to alcohol of Actinomyces sp. from Nocardia sp. or other decolorization weakly or partially acid fast organisms. ○ Gram (-) bacteria: those with thin peptidoglycan This is applied to sputum, other fluids, and tissue cell walls and outer membrane that can be samples when acid-fast bacilli are suspected. dissolved with alcohol or acetone. Identification of the pink or red acid-fast bacilli Cellular morphology and gram stain characteristics often against the blue background of the counterstain can be used to categorize stained organism into groups requires a trained eye as few acid-fast bacilli may be such as Streptococci, Staphylococci, and Clostridia detected in the smear even after centrifugation. Alternative method: AURAMINE-RHODAMINE COMBINATION FLUORESCENT DYE TECHNIQUE, STEPS IN GRAM STAINING requiring an immunofluorescence microscope. ○ The results are faster because of the contrast STEPS GRAM (+) GRAM (-) provided between the red fluorescent organisms versus the black background. Kills bacteria ○ Although this is a more sensitive test, it is less Makes sure that bacteria will specific than the acid fast staining technique. Heat Fixation NOT be washed or rinsed off during staining STEPS IN ACID FAST STAINING Crystal Violet Stains all bacteria on the slide purple STEPS ACID-FAST (+) ACID FAST (-) Addition of mordant (iodine) Iodine Iodine locks crystal violet stain Prepare smear on slides Heat fixation Treatment and gives the stain better Kill bacteria via heat fixation penetration and diffusion Carbolfuchsin Slides are heated to drive the stain Washes the stain red staining into the cells, staining all cells red from the thin Thick cell-walled Organisms will Carbolfuchsin cell-walled Decolourization organisms will Decolourization retain red will be organisms, remain purple using acid carbol-fuschin washed out, cells becoming colourless alcohol dye, remaining will appear red colourless Purple-stained organisms do Carbol-fuschin Thin cell-walled Counterstaining dye will outweigh Cells will take up Counterstaining NOT take up organisms are by methylene methylene blue, methylene blue, by Safranin safranin, stained pink blue cells will remain will appear blue remaining purple red Gram-staining. Acid-fast stain. Microbiology - Mod 1 Principles of Diagnostic Microbiology 4 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited. B. GROWTH AND CULTIVATION ○ At the onset of illness: acute phase sample Required for definitive identification and ○ 10-14 days later: convalescent phase sample characterization Could either be labeled or non-labeled reagent assays Usually the most sensitive and specific means of Have limitations in sensitivity because they have to diagnosis form large antigen-antibody complexes for detection Cause of an infection is confirmed by isolating and For rapid detection of bacteria and viruses culturing microorganisms either in artificial media or in living hosts. 3. GENOTYPIC IDENTIFICATION Isolation of infectious agents frequently requires Genotypic identification techniques for the detection and specialized media or artificial media that support quantification of specific DNA and RNA base sequences bacterial growth in vitro. in clinical specimens have become powerful tools for the ○ Non-selective or non-inhibitory media permit the diagnosis of bacterial, viral, parasitic, and fungal growth of many microorganisms. infections. ○ Selective media contain inhibitory substances that Genetic probes identify genus or species specific permit isolation of specific types of microorganisms. DNA or RNA sequences. Pathogenic viral agents are often sought by culture Nucleic acid tests are used for 4 purposes: when: ○ Detecting or quantifying specific pathogens in ○ Presence of serum antibodies is not a criterion clinical specimens for active infection. ○ Identifying organisms, usually bacteria, that are ○ Serologic diagnosis is not practical. difficult to identify by conventional methods ○ Immunoassays have inadequate sensitivity. ○ Determining whether 2 or more isolates of the same pathogen are closely related, i.e. belonging C. ANALYSIS OF CULTIVATED ORGANISMS to the same clone or strain ○ Predicting the sensitivity of organisms, typically 1. PHENOTYPIC IDENTIFICATION OF BACTERIA viruses, to chemotherapeutic agents Once bacteria are isolated, characteristics (e.g. colony size, colour, hemolytic reaction, odour, microscopic IV. ANTIBIOTIC SUSCEPTIBILITY ASSAYS appearance) that are readily detectable after growth Microorganisms, particularly, bacteria are tested in vitro on agar media may suggest the species. to determine whether they are susceptible to But, definitive identification requires additional tests antimicrobial agents. such as growth characteristics under various A principal responsibility of a clinical microbiology conditions, utilization of carbohydrates and other laboratory is to determine which antimicrobial agent substrates, and enzymatic activity. inhibits a specific isolate. IDENTIFICATION OF VIRUSES ○ Neutralization and serologic identification A. DIFFUSION TESTS (AGAR) Neutralize infectivity by mixing isolate with Zone diameters are interpreted as signifying specific antibody to known viruses before susceptibility, intermediate susceptibility, or resistance to inoculation into cultures each antimicrobial agent tested. Inhibition of expected viral effects on the cell structure such as cytopathic effects (CPE) or 1. KIRBY-BAUER DISC DIFFUSION METHOD hemagglutination is evidence for virus A commonly used diffusion test procedure. ○ Cytology and histology The in vitro response of the bacteria to a Intranuclear inclusions standardized antibiotic containing disk has been Cytoplasmic inclusions correlated to the clinical response of patients given the Cell fusion with multinucleated giant cells drugs. ○ Electron microscopy Involves the placement of paper discs containing antibiotics on an agar surface inoculated with the 2. IMMUNOCHEMICAL METHODS bacterial strain to be tested Infection may be diagnosed by an antibody response Measurements of zones of growth inhibition after to the infecting microorganism. incubation. Specially useful when the suspected microbial agent either cannot be isolated in culture or by any known method, or can be isolated in culture only with great difficulty Techniques for the detection and quantitation of antigens/antibodies ○ Measurement of serum antibodies provides an indirect marker for past or current infection with a specific viral agent or other pathogens. ○ The biologic signals are usually IgM or IgG antibodies directed at surface expressed antigen. A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis. The determination of an antibody response is a measure of current immunity and is important in the case of viral agents for which there are vaccines. Quantitative serologic assays to detect increases in antibody titers (i.e. level of antibodies in a blood sample) Kirby-Bauer Disc Diffusion Method. most often employ paired serum samples obtained: Microbiology - Mod 1 Principles of Diagnostic Microbiology 5 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited. B. DILUTION TESTS Provides a quantitative estimate of susceptibility to an antibiotic Involves inoculation of the test strain of bacteria into a series of broth cultures or agar plates with increasing concentrations of antibiotics 1. MINIMUM INHIBITORY CONCENTRATION (MIC) Lowest or minimal concentration of antibiotics that INHIBITS visual microbial growth Can be given a categorical interpretation of susceptible, intermediate, or resistant, so it is more widely used than the MBC 2. MINIMUM BACTERICIDAL CONCENTRATION (MBC) AKA Minimum Lethal Concentration Lowest concentration of the chemotherapeutic agent that results in NO growth of the subcultures Lowest or minimal concentration of antimicrobial agent that KILLS at least 99.9% of the inoculum used for the test ANTIBIOTIC SENSITIVITY Microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally SUSCEPTIBLE recommended dose of the antimicrobial agent Implies that infection by microorganism may be treated with antimicrobial agent Indicates that the microorganism is resistant to concentrations of the antimicrobial agents that can be attained with normal doses RESISTANT Implies that infection by microorganism could not successfully treated by antimicrobial agent Microbiology - Mod 1 Principles of Diagnostic Microbiology 6 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.  APPENDIX Flow of Procedures in the Microbiologic Diagnosis of Disease in a Patient Suspected With Infection. Microbiology - Mod 1 Principles of Diagnostic Microbiology 7 of 7 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.