Subject: Microbiology Topic: Principles of Diagnostic Microbiology Lecturer: Dr.

Teresa Barzaga Date of Lecture:06/23/2011 Transcriptionist: Group 5 Pages: 19

DIAGNOSTIC MICROBIOLOGY Encompasses the characterization of thousands of agents that cause or are associated with disease Concerned with the etiologic diagnosis of infection

-

-

-

Prepare and update manuals regarding appropriate specimen collection and handling Save patients money by providing accurate diagnosis in a timely manner using the most cost-effective techniques

SERVICES OF A MICROBIOLOGY LABORATORY: Provide relevant information Establish guidelines for proper specimen collection (MOST IMPORTANT: good quality specimen) Timely specimen transport Identification of pathogens Susceptibility testing (to know what drug to prescribe) Rapid reporting test result

FUNCTIONS OF A MICROBIOLOGY LABORATORY: 1. Etiologic diagnosis of an infectious disease a. Isolation and identification of an infectious agent b. Demonstration of an immunologic response 2. Set guidelines for rational use of antimicrobials GUIDELINES IN SELECTION AND COLLECTION OF CLINICAL SPECIMEN 1. COLLECTION - From active site of infection o If suspected CNS infection, collect CSF not sputum - Timing o During acute stage of disease, before giving antibiotics o Antibiotics can kill the organisms, giving false negative results - Amount o Adequate for culture

-

RESPONSIBILITY OF MICROBIOLOGISTS TO PATIENT AND CLINICIAN Identify and control nosocomial pathogens - Track resistant organisms - Coordinate the antimicrobial agents tested with the Therapeutics Committee o Therapeutics Committee decide what drugs should be available in the pharmacy -

1

SY 2011-2012

& Antibiotic Susceptibility Immunoassays

o Sputum: 5ml or 1 teaspoon - Proper tools and containers o Urine, stool, CSF: Use sterile containers without transport or preservative - Preservatives can kill the microorganisms o Tissue: Use sterile, dry, widemouth, screwcapped bottle or plastic container without water or saline o Blood:Sterile tubes containing broth or anticoagulant  STERILE SWABS - Most convenient and most commonly used tool for specimen collection - Provides the poorest conditions for survival and can only absorb a small volume of inflammatory exudates - SWABS LIMIT VOLUME AND SURVIVAL 2. DELIVERY, TRANSPORT, PROCESSING - Immediately - Viability may be lost if specimen is delayed - Transport media stabilize conditions and prevent drying  Minimizes the effects of delay between specimen collection and laboratory processing - Example: Stuart, Amies and Cary Blair transport media 3. STORAGE - Appropriate conditions - All specimens can be kept at room temperature EXCEPT: o 4°C

To prevent overgrowth of organism present in the specimen  Urine  Stool  Viral specimen  Sputum  Swabs  Foreign devices  Gastric biopsies  Rectal swabs  Stool for culture o 37°C  CSF o -20°C  Serum serologic studies (stored up to one week) o -70°C  Tissues or specimens for long-term storage o Use a low temperature or deep freezer o Not all freezers have the same temperature o Ordinary household freezer is about -20°C o Ultra low freezer is about -70° C o Liquid nitrogen freezer is about -196°Cfor very long storage purposes.

-

Quality of the specimen is crucial Types of specimens FLUIDS Blood Urine CSF

2

-

Secretion Aspirate Cavity fluids From cystic cavities

The Specimen A. DIRECT TISSUE OR FLUID SAMPLES - Collected from normally sterile tissues (lung, liver) and body fluids (CSF, blood) Methods by which it can be obtained are: 1. Needle aspiration 2. Surgical biopsy (very sterile) *Specimen collected contains only the pathogens - Positive findings: diagnostic - Negative findings: can exclude - Infection at suspected site *DIRECT SAMPLES GIVES HIGHEST QUALITY B. INDIRECT SAMPLE Pathogen is localized in sterile and must pass through a site containing normal flora in order to be collected *SPECIMEN CONTAINS THE PATHOGEN BUT IS CONTAMINATED WITH NON PATHOGENIC FLORA SPECIMENS OF INFLAMMATORY EXUDATES (expectorated sputum, voided urine) that have passed through sites colonized with normal flora. For example, urine is sterile when it s in the urinary bladder but when patient voids, it passes through urinary tract and passes through urethral orifice and causes contamination. Sputum is sterile when it is in the lungs, but when it is expectorated to the upper respiratory tract to the mouth, it gets contaminated because

ABSCESSES BONE MARROW SCRAPINGS Corneal Skin

SOLID/SEMI-SOLID Tissues Hair and nails Swabs Stool

FOREIGN BODIES IUD (intrauterine device) I.V. catheters o Common source of organism because when we do intravenous administration, we puncture the skin, and if it is not properly cleaned, then you introduce organism on the skin into the blood stream and cause infection. o We recommend time for change of IV catheter in 48 hrs / whenever there is a sign of infection. Pins Prosthatic valves

-

-

-

3

the mouth contains millions of organism. *Degree of contamination is related to the skill with which the normal flora site was bypassed"in specimen collection Bypassing normal flora requires extra effort o In obtaining sterile urine, there issuprapubic aspiration: Don t allow patient to void so don t allow the specimen to pass through the urethral meatus but puncture directly through suprapubic area on the skin with syringe and aspirate but that s very invasive unless in an emergency, avoid doing that. o For sputum, some doctors use broncho-alveolar lavage or endotracheal aspiration; but still, this is very invasive Result require interpretive evaluation of contamination

peritoneal cavity and cause the disease peritonitis *PATHOGENS AND NON PATHOGENIC FLORA ARE MIXED AT THE SITE OF INFECTION *Both are collected and the nonpathogens are either inhibited by use of selective culture methods or discounted in interpretation of culture results. D. FOR VIRAL DIAGNOSIS Easier: essential little normal viral to confuse interpretation - Lack of normal viral flora simplifies interpretation y Allows selection guided by the knowledge of which sites are most likely to yield the suspected etiologic agent MUST KNOW (*LIKELY TO BE ASKED IN EXAM) SAMPLING SITES AND NORMAL FLORA NORMALLY STERILES SITES (no flora) - Blood and bone marrow - Peritoneal and pulmonary cavity - Posterior urethra - Urinary bladder - Accessory nasal sinuses - Trachea, bronchi, alveoli - CSF - Kidney, prostate - Uterus - Larynx - Middle and inner ear SITES WITH NORMAL COMMENSAL FLORA: - Mouth - Nose

-

C. SAMPLES FROM NORMAL FLORA SITES Primary site of infection: in an area known to be colonized with many organisms (pharynx and large intestines) o The large intestine contains millions of gram negative organisms. o E.coli, which are called resident flora, don t cause any disease in the large intestine but once the large intestine is ruptured; bacteria spills out into sterile

4

-

Skin Female genital tract External genitalia External ear canal Throat Oropharynx Gastrointestinal tract Urethra Eye

CRITERIA FOR REJECTION OF SPECIMEN 1. Patient information on requisition slip a. Erroneous entry, incomplete data - PhilHealth nowadays has become very strict about the patients information (age, date of birth etc.) - Sometimes, there are patients who have exactly the same names! Thus, the samples collected might be interchanged. 2. Container a. Improper b. Leaking - Improper and leaking containers are immediately rejected. 3. Specimen a. Improperly collected - If this occurs, the personnel in charge (medtechs) are required to collect a new sample. This applies especially on tissues placed in fixatives (stabilizing or preserving agents). b. Dried up/dried swab c. Placed in a fixative d. Pooled specimen (24 hour) - A pooled specimen is a sputum sample. - In the past, pooling of sputum samples is advised because it is believed that the more pool samples that you have, the greater the chance of isolating

METHODS FOR CIRCUMVENTING INDIGENOUS FLORA 1. ANTISEPSIS a. 1-2minutes contact time with a disinfectant (70% alcohol or betadine) especially for blood culture 2. By-pass areas with normal flora (invasive) a. Transtracheal aspiration b. Open biopsy c. Percutaneous needle biopsy d. Suprapubic aspiration of urine 3. Culture specifically for organisms known to be etiologic a. Agents (e.g. Group A streptococci in throat) 4. Quantities culture results (e.g. quantitative urine cultures) 5. Decontamination for mycobacterial cultures a. 4-10% NaOH or hypochlorite solution b. e.g. We use this for TB mycobacterial because sputum is contaminated in mouth 6. Selective media a. Salmonella-Shigella agar b. TCBS thiosulfate-citrate bile salt solution (for Vibrio cholera)

5

or obtaining a positive sample. (e.g. Mycobacterium tuberculosis) - Nowadays, pooling samples is not advised because it was realized that the more positive results that you obtain, there is also a greater chance of contamination. - Three early morning sputum samples are the best samples that can be collected (e.g. for TB cultures). e. Insufficient quantity - Quantity is a very important criterion for collection of specimen. 4. Transport a. Improper temperature b. Inappropriate transport container c. Delay of collection and arrival in the laboratory - It is a daily routine to stamp the specimen. This is done to avoid confusion and to confirm the actual time when the specimen was obtained. 5. Processing a. Produce information of questionable medical value

- Agar media III. Analysis of Cultivated Organisms

I. DIRECT EXAMINATION 1. Microscopy - Most commonly used method for direct examination

Bright field microscope - Visible light passed thru specimen series of lenses which reflects light in a manner that result in magnification of organisms present. - Total magnification is a product of the lenses used (objective lens + eyepiece).

Phase-contrast microscopy translates differences in phase within the specimen into differences in light intensities resulting in contrast among objects within the specimen. - Allows observation of viable organisms - Identification of important fungi grown in the culture.

FLOW OF PROCEDURES IN LABORATORY DIAGNOSIS OF INFECTIOUS DISEASES (step by step) I. II. Direct Examination Growth and Cultivation

Fluorescent microscopy when dye molecules (fluorochromes) which have been raised to a higher energy level after absorbing UV light returns to the lower energy state, excess energy is

6

released in the form of viscible (fluorescent) light. - In the example mentioned, the acid fast bacilli stained yellow against a black background.

protozoa in blood and tissues.

Stained smears (Bacteria must be stained!!) i. Simple stains uses single stain and all cells and structure stain same color. *Positive stain is attracted to cells which take on the color. *Negative stain is repelled by the cells and background takes on color.

Darkfield Microscopy - Condenser doesn t allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle. - Used to detect spirochetes (T. pallidum)

Electron microscope this uses electrons instead of photons (light). The electrons are focused by electromagnetic field and form an image on a fluorescent screen. Have 2 types: Scanning Transmission

ii. Differential stains - specific types of organisms or structures exhibit different staining reactions that can be distinguished by different colors. Gram stain principle: numerous teichoic acid cross linkages contribute to the ability of gram (+) organisms to resist alcohol decolorization.

2. Staining techniques a. Smear preparation - Apply sufficient material to slide. Wet Mounts Direct fecal smear, KOH mount, NSS mount, hanging drop. It is used to demonstrate: blood cells and microbes in fluid specimens, cysts, eggs and parasites, fungi in skin,

-

7

1. Heat fixation allows the bacteria to stick into the slide to avoid from being washed away. 2. Crystal violet as the primary stain will stain all of the organisms blue or violet gram(+) or gram(-), INITIALLY. 3. Iodine is a mordant (an element which aids the chemical reaction that takes place between the dye and the fiber so that the dye is absorbed). 4. Decolorization is done by adding alcohol.Gram (+) will maintain the blue/violet color and gram (-) will be colorless. 5. Applying safranin on a gram(+)stain will retain its blue color while applying it on a gram ( ) one will produce a red color. * Gram positive bacteria retain purple iodine dye complexes. * Gram negative bacteria do not retain complexes when decolorized. * Properly decolorized background should be red. * Gram reaction plus morphology guide clinical decisions e.g. giving antibiotics. there are some antibiotics which are selective only on g + or g bacteria.

- Weak initial staining and strong retention is related to the HIGH LIPID CONTENT of the mycobacterial cell wall.

*This is just the same as gram staining EXCEPT that it uses carbolfuchsin red as the primary stain, the counter stain is methylene blue and it doesn t use iodine (mordant in gram staining).

Endospore stain permits differentiation of endospore from vegetative cell.

*Herpes viruses under the electron microscope

Acid fast stain Principle: bacteria whose cell wall contain mycolic acids render cells resistant to decolorization even with acid alcohol decolorizers.

8

o

*Summary of the different microscopic methods

II. GROWTH AND CULTIVATION: required for definitive identification and characterization; most sensitive and specific way of diagnosis 1. Grow and isolate all bacteria in an infection 2. Etiological agents are identified 3. Allow identification and characterization 4. Assess susceptibility of the pathogen to the antibiotics 5. Direct observation will just provide a presumptive diagnosis, like when doing gram staining

Principles A. Nutritional requirements o Phases of growth media  Liquid (broth) bacterial growth is indicated by change in appearance from clear to turbid y Large numbers of bacteria in the broth produce turbidity  Solid (agar)

Agar is a convenient gelling agent for solid media Media classification and function  Enrichment media enhance growth of a particular bacterial pathogen from a mixture of organisms using nutrient specificity (e.g. buffer charcoal yeast extract (BCYE) for Legionella pneumophilia; upon discretion of clinician)  Supportive media contain nutrients that support the growth of most non-fastidious organisms (e.g. xylose lysine deoxycholate agar: for the growth of Shigella only)  Selective media contain one or more agents inhibiting all organisms except what is being searched for. (e.g. Phenyl ethyl alcohol agar, which allows only the enterococci to grow; Deoxycholate-citrate agar: inhibits E. coli and allows salmonella,shigella.  Differential media use factors that allow colonies of one bacterial species to exhibit metabolic or cultural characteristics that will distinguish them from other bacteria on the same plate. (e.g. MacConkey agar and eosin methylene blue (EMB) agar: used to grow gram (-) organisms  Special media - only for specific organisms y Sabouraud dextrose agar: fungi; the low pH y

9

encourages the growth of most fungi y Lowenstin Jensen (LJ) media: favor growth of mycobacteria; contain glycerol and malachite green (inhibitory for all other bacteria except mycobacteria) y Bordet-Gengou agar: for Bordetella pertussis y Fletcher medium: for Leptospira B. Environmental requirements o Oxygen and CO2 availability  Aerobic: thrive withO2  Facultative anaerobes: thrive with or without O2  Anaerobes: do not thrive with O2 Temp: Optimum 35°C-37°C pH 6.5-7.5 Moisture of medium and atmosphere is needed

surfaces of the growth with contaminated organisms c. Isolation i. Streak plate a loopful of bacterial cells is streaked across the surface of a sterile agar plate that contains media

*Last quarter (upper left) of the plate wherein you see individual bacterial colonies. In primary streak, they re all together, hard to identify. From the last streaking, there are individual colonies.

o o o

Process of bacterial cultivation expose media A. Establishing a pure culture a. Sterilization i. Exposure to elevate temperature ii. Toxic iii. Radiation iv. Filtration 0.20-0.45 v. Dry heat vi. Autoclaving: resulting in a particular smell in the laboratories b. Aseptic techniques i. Contact of the pure culture, sterile medium, and sterile

ii. Spread plate a drop of bacterial suspension is placed on the center of an agar plate and spread over the surface iii. Pour plate bacterial suspensions are added to tubes containing cooled melted agar (42°C-45°C); mixed and poured onto sterile petri dishes d. Maintaining and preserving pure cultures i. Subculture periodically 1. Especially for control strains: organisms to which the bacterial growth is compared a. American type culture collection b. Controls are subjected to culture sensitivity: do biochemical and sensitivity tests to know expected reaction 2. At least once a week or every 3 days

10

ii. Refrigeration at 0° C- 5° C iii. Freezing in liquid nitrogen (-196° C) iv. Lyophilization (freeze-drying) v. ATCC (American type culture collection)

III. ANALYSIS OF CULTURED ORGANISMS Principles of identification 1. Phenotypic identification of bacteria based on observable physical and metabolic characteristics of the bacteria A. Macroscopic morphology

a) Accomplished by the use of agar media supplemented by inhibitory substances or antibiotics, measuring directly the organism s resistance to antimicrobial agents that may be used to treat the infection D. Microscopic morphology and staining characteristics a) Gram staining: usually the first step in the identification scheme i. Divide bacteria to four: 1. Gram (+) cocci 2. Gram ( ) cocci 3. Gram (+) bacilli 4. Gram ( ) bacilli E. Nutritional requirements F. Enzymatic capability Types of enzyme-based tests; she said that this needs to be memorized) A. Single enzyme test i. Catalase test: for gram (+) identification 1. Positive test: rapid production of bubbles when the bacterial growth is mixed with H2O2 solution ii. Oxidase test: gram ( ) identification 1. Positive test: development of dark purple color when reagent substrate is inoculated with bacterial growth iii. Indole test: used to presumptively identify E. coli 1. Positive test: development of a blue green color when a filter paper disk impregnated with test substrate(indole) is inoculated with bacterial growth

a) Colony size: measured in mm or described as pinpoint, small, medium, or large b) Color c) Elevation, margins, shape d) Colony appearance: dull, glistening, opaque, or transparent e) Changes in agar media resulting from bacterial growth: whether there is presence of hemolysis on blood agar plates f) Change in pH g) Odor i. Proteus species have a particular odor B. Environmental requirements for growth C. Resistance or susceptibility

11

iv. Urease test: helps to identify Proteus species, Helicobacter pylori 1. Positive: development of a bright pink or red color when test substrate(urease) is inoculated with bacterial growth

- Can be seen as cell rounding or cell fusion - eg. From spindle-shape to being round --- that is positive CPE 2. Production of hemagglutinins *May be present on the infected cell membranes, in culture media as a result of release of free, hemaggluntinatingvirions from the cells *Addition of erythrocytes results in adherence to cell surfaces (HEMADSORPTION) *Allantoic and amniotic fluid is used to test the presence of hemagglutinins *Presence of hemagglutininsis indicative of presence of virus in the sample. C. In Vivo Isolation Methods 1. Embryonated hen s egg - Inoculation of specimen on egg membrane --- incubation 2. Animal inoculation - Uses suckling mice: viral replication is seen as development of illness D. Viral Identification 1. Neutralization and serologic identification *Neutralize infectivity by mixing isolate with specific antibody to known viruses before inoculation into viruses before inoculation into

Test for Presence of Metabolic Pathways May involve several interactive enzymes End product from the interactions are measured a. Carbohydrate oxidation and fermentation b. Amino acid degradation c. Single substrate utilizations ISOLATION AND IDENTIFICATION OF VIRUSES A. Cell and Organ Culture - Living cell cultures that can support replication are the primary means of isolating pathogenic viruses y CELL CULTURE DERIVED FROM HUMAN OR ANIMAL TISSUES ARE USED TO ISOLATE VIRUSES - Cells are derived from a tissue fragment (explant) or by dispersal with proteolytic enzymes such as trypsin B. Detection of Viral Growth 1. CPE (cytopathic effect) *VIRAL CPE IS DUE TO MORPHOLOGIC CHANGES OR CELL DEATH - As viruses replicate in cells, there is production of alterations in cellular morphology (or cell death) which are seen by light microscopy

12

cultures --- inhibition of expected viral effects on the cell culture such as CPE or hemagglutination is evidence for the virus 2. Cytology and histology *Intranuclear inclusions; cytoplasmic inclusions; cell fusion with multinucleated giant cells 3. EM

Advantage: yields result faster

CLASSES OF IMMUNOASSAYS A. Precipitin immunoassays - Reaction that forms insoluble lattice when large complexes of antigen antibody are combined - Can provide qualitative/semiquantitative analysis

LIMITATIONS OF PHENOTYPIC IDENTIFICATION: 1. Inability to grow certain fastidious organisms 2. Inability to maintain viability of certain pathogens during transport 3. Extensive delay in cultivation and identification of slowly growing pathogens 4. Lack of reliable methods to identify certain organisms grown in vitro 5. Use of considerable time and resources in establishing the presence and identity of pathogens II. IMMUNOCHEMICAL METHODS Techniques for the detection and quantitation of antigens/antibodies Could either be labeled or non-labeled reagent assays Have limitations in sensitivity because they have to form large antigenantibody complexes for detection For rapid detection of bacteria, fungi, parasites, viruses It is a non-cultural method which only needs serum

FACTORS AFFECTING PRECIPITATION REACTIONS: Relative proportion of reactants *On the pattern of precipitin formation, it can be noted that there is a point of maximum precipitation Temperature pH 6.0 to 7.5 Ionic strength of the medium

CLINICAL LAB APPLICATIONS OF PRECIPITININ REACTIONS 1. Single immunodiffusion - Antigen diffuses from overlay into agar containing antibody forming precipitin line at point of equivalence - Can be carried out in test tubes, petri dishes, slides - In this method, antibodies are measured 2. Ouchteriony double diffusion

-

13

-

Patient specimen containing antigen (in a well) +antibody directed against the antigen (in an adjacent well) --- Ag and Ab will diffuse toward each other in 18-24hrs --- visible precipitin band at the meeting point (zone of equivalence)

o

o

Reagin (Ab-like protein produced by patients infected with T. pallidum) binds to the test antigen (cardiolipin-lecithincoated cholesterol particles), causing the particles to flocculate Not highly specific thus replaced by RPR

3. Electroimmunodiffusion Variation of double immunodiffusion using an electric current to augment the diffusion(rapid completion of assay) Use of electric current makes the reaction faster

b. RPR (rapid plasma regain) o o Most commonly used nowadays Similar to VDRL but more specific as a screening test for treponemapallidum/syphil lis Test consists of positive and negative controls, the reaction card, prepared antigen suspension Antigen also contains charcoal particles to allow macroscopically visible flocculation Sera tested without heating Not recommended for testing CSF

-

o 4. Immunoelectrophoresis Couples electrophoretic separation with the 2 dimensional immunodiffusion

o

5. Radial Immunodiffusion Quantitative technique Same principle as in single immunodiffusion but uses antigen standards of known amount o o

6. Flocculation test The precipitin end product forms macroscopically or microscopically visible clumps a. VDRL (Venereal Disease Research Laboratory)

B. Particle Agglutination Detects antigen via agglutination (clumping) of an artificial carrier particle such as a latex bead with antibody bound to its surface 1. Latex agglutination o Antigen is present in a specimen being tested bonds to the

14

combining sites of the antibody exposed on the surfaces of the latex beads --- forms crosslinked aggregates of latex beads and antigens 2. Hemagglutination (indirect or passive) o Uses treated animal red blood cells as carriers of antigen for agglutination tests a. Microagglutination test for antibody to Treponemapallidum (MHA-TP) b. Hemagglutinationtreponemal test for syphilis (HATTS) c. Rubella indirect hemagglutination tests d. Passive hemagglutination tests for antibody to extracellular antigens of streptococci 3. Coagglutination

o

o

Includes 2-site immunometric sandwich assays and indirect assays to measure antibodies Enzyme reaction product concentration directly proportional to concentration of standard/analyte antigen

ADVANTAGES OF EIA 1. Reagents are relatively cheap and have long shelf life 2. Sensitive assays can be developed by amplification effect of enzymes 3. Formation of a colored end product allows direct observation of the reaction or automated spectrophotometric reading 4. No radiation hazards 5. Equipment is inexpensive and is widely available

DISADVANTAGES OF EIA 1. Measurement of enzyme activity can be more complex 2. Enzyme activity may be affected by plasma constituents

C. Enzyme immunoassays Enzymes used in EIA tests: Basic test consists of antibodies bonded to enzymes which remain able to catalyze a reaction yielding a visually discernible end product while attached to the antibodies (change in color might be observed) Antibody-binding sites remain free to react with their specific antigen 1. Horseradish peroxidase 2. Alkaline phosphatase 3. Avidin-biotin

-

1. Competitive assays (analyte assays) 2. Noncompetitive/immunometric sandwich assays (reactant excess)

D. Immunofluorescent assays - Uses fluorescent dyes that can be covalently attached to antibody molecules and made visible by ultraviolet light in the fluorescent microscope - Used for detecting antibodies

15

-

Antigens are immobilized and fixed onto glass slides with formalin, ethanol or acetone --- addition of conjugated to fluorescent dyes --use fluorescent microscope

interpreted as a definitive identification of the organism

METHODS OF NUCLEIC ACID ANALYSIS A. Agarose gel electrophoresis - Separates DNA fragments or plasmids - (ubiquitousextrachromosomal elements composed of doublestranded DNA that is typically circular) based on size *Speed of migration depends on size: small molecules move faster and appear at bottom of gel B. Restriction endonuclease digestion Refines electrophoretic analysis of DNA Restriction endonuclease: enzymes that recognize specific nucleotide sequences on DNA molecules and digest (cut) them at all sites at which the sequence appears

2 TYPES: 1. Direct (DFA)  FITC is conjugated directly to the specific antibody  Faster than IFA 2. Indirect (IFA)  Antigen-specific antibody is unlabeled and a second antibody (usually raised against the animal species from which the antigen-specific antibody was harvested) is conjugated to the FITC  More sensitive than DFA

E. Chemiluminescent assays Uses chemiluminescence-generating molecules as labels (luminal derivatives, acridinium esters, nitrophenyl oxalate derivatives) Assay principle and method does not differ from that of heterogenous immunoassays, radioimmunoassays, and fluorescent immunoassays

C. DNA hybridization Allows DNA from different sources to combine Opened DNA helix leaves singlestranded DNA exposing nucleotide bases which become available to interact with other single-stranded nucleic acid molecules

-

GENOTYPIC IDENTIFICATION Characterization of some portions of the genome using molecular techniques for DNA or RNA analysis The presence of a specific gene or a particular nucleic acid sequence is D. Polymerase Chain Reaction Amplification technique that allows the detection and selective replication of a targeted portion of the genome Faster way used in the laboratory

-

-

16

Antibiotic Susceptibility Assay Methods of Nucleic Acid Analysis Polymerase chain reaction (PCR) Amplification technique that allows detection and selective replication of a targeted portion of a genome Faster way to do identification Allows study of organisms that cannot be cultured General Indications: 1. Isolation of organisms with unpredictable susceptibility to antimicrobial agents (e.g. Staphylococci, Enterobacteria, Pseudomonas) 2. Isolation of organisms of clinical significance (e.g. isolates from normally sterile sources, wounds and abscesses, and urine if present in significant numbers) Importance: a) When an antibiotic has a narrow therapeutic index a. The concentration required for successful treatment and the concentration that is toxic to the patient are not very different b) When the normal route of excretion of antibiotic is impaired a. Patients with renal failure c) When the absorption of the antibiotic is uncertain a. Particularly after oral administration d) In neonates with serious infections e) To ascertain concentrations in sites of infection into which penetration of antibiotics is irregular or unknown a. In cerebrospinal fluid f) In patients receiving prolonged therapy for serious infections a. Some patients who have been on antibiotics for a long time -> do testing to see if patient is sensitive/ resistant to treatment b. See if organism infecting patient is still sensitive to antimicrobial you are prescribing g) In patients who fail to respond to apparently appropriate therapy

-

Application of Nucleic Acid Methods to Infectious Diseases: When do we use Nucleic Acid methods for diagnosis? 1.Bacterial or Viral genomic sizing Used in research, not usually done in the laboratory Number and size of plasmids differentiates strains Endonuclease digestion of plasmids refines their comparison

2.DNA Probes Probe: a fragment of DNA that has been cloned or recovered from a genomic or plasmid source Can detect DNA of pathogens directly in clinical specimens

-

3. Polymerase Chain Reaction Allows study of organisms that cannot be cultured

*See Textbook for Route from Patient to Microbial Diagnosis

17

a. Patient doesn t seem to be getting well, patient may be resistant, do testing h) To check on patient compliance a. If patient is really taking prescription given

containing the antibiotics are applied, followed by overnight incubation then zones of inhibition around each antibiotic disk are observed *** Zone diameters are interpreted as signifying susceptibility, intermediate susceptibility, or resistance to each antimicrobial agent tested 2. Dilution Tests preferred by clinicians due to MIC and MBC - Provides a quantitative estimate of susceptibility to an a antibiotic - Serial dilutions of the test antibiotics that are prepared in broth or agar medium, then a suspension of test organism is inoculated then incubated overnight

Antibiotic Susceptibility Assay Not necessary when the organism is known to be susceptible to the primary drug of choice o E.g., In the province, there s no lab that can do testing, just give medication Usually performed with organisms that grow rapidly and well on artificial media

-

Susceptibility test results aid in: 1. Adjustment of initial dosage given [add more, etc.] 2. Modification of existing therapy [ex. If not responding] 2 Categories of Dilution Tests Minimum Inhibitory Concentration [MIC] - Lowest or minimal concentration of the antimicrobial agent that is required to inhibit the growth of a microorganism - Recorded as the highest dilution in which there is no macroscopic growth - Expressed in micrograms/milliliter

Selection of agents to be tested Begins with coordination between Pharmacy and Therapeutics Committee of Hospital In accordance with the National Committee for Clinical Laboratory Standards guidelines [gram (+) = use gram (+) antimicrobial]

-

Minimum Bactericidal Concentration or Minimum Lethal Concentration [MBC] - Lowest or minimal concentration of antimicrobial agent that kills at least 99.9% of the inoculum used for the test - Used in the evaluation of a new antimicrobial agent

2 Categories 1. Diffusion Tests easier - Isolate is seeded over the entire surface of an agar plate (streak) and filter paper disks

18

Factors affecting tests: 1. Composition of medium - Use Mueller-Hinton medium for testing aerobic and facultative bacteria a. Demonstrates good batch to batch reproducibility b. Low in sulfonamide, Trimethoprim and tetracycline inhibitors c. Supports the growth of most nonfastidious pathogens

McFarland s Standard y y Tube that contains barium sulfate (BaSO4) Turbid liquid used to compare turbidity of organism that you will put into agar plate Should be approximately the same, compared visually Signal to start planting into agar plate, putting filter paper

y 2. Medium-related factors a. Agar depth should be about 4mm i. Too deep: false-resistant results ii. <4mm: falsely susceptible b. Media containing excessive amounts of thymidine i. Can reverse the inhibitory effects of sulfonamides and trimethoprim: less distinct zones of inhibition 3. Inoculum a. Inoculum size approx. 5x105 CFU/ml in broth dilution tests b. Method of preparation: should be prepared directly from overnight growth on an agar medium y

4. Incubation a. General recommendations: 16-18 hours in ambient air at 35oC b. Exceptions i. S. pneumoniae, H. influenza, N. gonorrhea ii. 5-7% CO2: 20-24 hours iii. Staphylococci and Enterococci: 24 hours iv. Anaerobes and yeasts: 48 hours ***END OF TRANSCRIPTION***

19

Sign up to vote on this title
UsefulNot useful