TRANSES — BACTE 2 (1).pdf

CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023...

CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 LESSON 5: SPECIMEN COLLECTION, HANDLING, TRANSPORT AND SPECIMEN PRESERVATION PRESERVATION 1. BORIC ACID A. Time of Collection - Urine B. Specimen Transport - Maintains the appropriate C. Specimen Storage Conditions colony counts D. Specimen Preservation E. Specimen Labeling 2. POLYVINYL ALCOHOL (PVA) OR BUFFERED FORMALIN F. Specimen Requisition - Stool G. Rejection of Unacceptable Specimen - Maintains the integrity of trophozoites and cysts of ova H. Appropriate Collection Techniques and parasite (O&P) Validity of any test result is primarily dependent on the quality of 3. STUART’S MEDIUM, AMIES MEDIUM, CARY AND BLAIR specimens received MEDIUM - Transport media / holding media TIME OF COLLECTION - Maintain the viability of microorganism without If possible, specimens should be collected during the acute supporting the growth of microorganism phase (early phase of illness) of infection and before the - No death and no overgrowth application of antimicrobial therapy 4. ANTICOAGULANTS SPECIMEN TRANSPORT - Prevents clotting of specimens (blood, bone marrow, ✓ Ideally, specimens should be sent to the laboratory as soon synovial fluid) as possible. In some cases, within 1-2 hours of collection, samples - Concentration and type of anticoagulant is important must be transported - Many organisms are inhibited ✓ Specimens must be placed in a leak-proof container ✓ If delay is anticipated, observe temperature requirements SODIUM POLYANETHOL SULFONATE (SPS) of suspected organism/s when transporting samples - 0.025% - 0.03% (others up to 0.05%) - Best anticoagulant for blood cultures SPECIMEN STORAGE CONDITIONS - Anti complementary, antiphagocytic ✓ Room (ambient) Temperature – 22°C - Interferes with actions of antimicrobials ✓ Refrigerator temperature – 4°C - Inhibits growth of some strains of Neisseria ✓ Body temperature (incubator) – 37°c - addition of 1.2% gelatin counteract SPS Inhibitory ✓ Freezer temperature – -20°C; -70°C if effect to some bacteria processing will be delayed more than 4 days HEPARIN - Viral cultures Room Temperature - Inhibits growth of gram (+) bacteria and yeasts Abscess Inner ear sample Bone Lesion Genital sample Tissue PRECAUTION: Wound Throat sample Preserved urine Use of the following anticoagulants are yet to be established in Body fluid Nasal sample microbiology therefore, must not be used: citrate - blue top SPECIMEN STORAGE CONDITIONS Ethylenediaminetetraacetic acid INCUBATOR - EDTA Cerebrospinal fluid for bacteria - lavender top Yellow top REFRIGERATOR a popular brand uses yellow top in two ways ▪ Catheter (IV) tips 1. SPS (Sodium Polyanethol Sulfonate) ▪ CSF for viruses 2. ACD (Trisodium Citrate Citric Acid Dextrose) ▪ Outer ear samples - not appropriate for microbiology ▪ Feces (unpreserved) ▪ Sputum Therefore, as a general rule… ▪ Urine(unpreserved) Do not refer to the use of tubes by color as different ▪ Feces for (C. Difficile toxin up to 3 days; >3 days store at -70°C) manufacturers have different combination of tube color and anticoagulant present Always refer to a tube with respect to the anticoagulant it carries CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 SPECIMEN LABELING 1. Patient’s name 2. Hospital number 3. Birthdate 4. Date and time of collection 5. Specimen source/type SPECIMEN REQUISITION 1. Patient’sname 2. Hospital number 3. Birth date/age 4. Specimen source/type 5. Date and time collected APPROPRIATE COLLECTION TECHNIQUES: BLOOD 6. Date and time received Vein selection (ante cubital fossa) 7. Examination Requested ▪ IV line: below IV line 8. Address/telephone number ▪ Avoid vascular shunts or catheters 9. Diagnosis ▪ Prosthetic devices are hard to decontaminate completely 10. Date of Admission 11. Current antimicrobial therapy 12. Ordering physician 13. Medico-legal cases: nature of incident, date and time of incident, place of incident 14. Sexually transmitted infections: civil status, occupation REJECTION OF UNACCEPTABLE SPECIMENS Specimen received for anaerobic culture from sites known to have Antisepsis anaerobes as part of the normal flora ▪ 70% alcohol ▪ Gastric washings ▪ Iodine (betadine) ▪ Urine other than suprapubic aspiration ▪ Iodine tincture ▪ Stool (except for C. Difficile – food poisoning) (iodine in alcohol) ▪ Vaginal secretions ▪ Chlorhexidine ▪ Oral/mouth/oropharyngeal specimens (except for deep tissues obtained during surgical procedure) Blood volume ▪ Swabs of ileostomy or colostomy sites 1-3 ml. - Pediatrics ▪ Superficial skin specimens 8-10 ml. - Adults Blood volume (Baron et. Al.) Number of Blood Cultures ▪ Periodicity of microorganism in bloodstream ▪ Random for some, continuous for some ▪ Two or three samples is a must ▪ Two or three sets is preferred ▪ As number of blood cultures is increased, pathogen recovery is increased CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 Timing of Collection ▪ Not so important Isolation of commensal microbial flora obtained from patients ▪ Organisms are released into blood stream at fairly constant rate suspected to be bacteremic ▪ Intermittent bacteria is unpredictable: ▪ Immunocompromised patients ✓ Two or three samples be spaced an hour apart ▪ Those having prosthetic devices Studies showed: APPROPRIATE COLLECTION TECHNIQUES:CSF No significant difference in the yield between multiple CSF FUNCTIONS simultaneous blood cultures or those obtained at intervals. ▪ cushioning and buoyancy for the bulk of the brain Conclusion: Blood volume collected is more critical than timing ▪ carries essential metabolites into the neural tissue and cleanses tissues of wastes as it circulates around the brain, ventricles and Blood Culture Techniques spinal cord. It produced by choroid plexus A. Conventional B. Instrument – based system B.1 BACTEC Systems (Becton Dickinson - Maryland) B.2 BACT/ALERT Microbial Detection Systems (Biomerieux-North Carolina) B.3. Versa TREK Systems (Thermo Scientific-Ohio) Blood Culture Media ▪ Trypticase Soy Broth ▪ Brain Heart Infusion Broth ▪ Thioglycolate Broth (Supplemented peptone) Columbia or Collection Brucella Broth 1. Skin antisepsis is applied before aspirating CSF 2. Lumbar puncture done by physician Interpretation of Blood Culture Results 3. Placed in sterile containers, screw-capped tube, without Growth of: additives Bacillus species Corynebacterium species, Propionibacterium 3 – 4 sterile containers are numbered sequentially according to acnes, Coagulase negative staphylococci the order in which they were collected ▪ Isolated in one of several cultures TUBE 1 - chemistry studies: glucose ▪ Bacillus anthracis must be ruled out before dismissing bacillus and protein, and immunology studies out before dismissing bacillus species as probable contaminants TUBE 2 - culture TUBE 3 - cell count and differential count Growth of multiple organisms from one of several cultures TUBE 4 – additional tests ▪ Polymicrobial bacteremia is Uncommon VOLUME 1 ml. – 5 ml The clinical presentation or course is not consistent with sepsis (up to 10 ml for mycobacteria and fungi) ▪ Physician-based not laboratory based criteria TRANSPORT The organism causing the infection at a primary site of infection ▪ CSF should be hand-delivered immediately to the laboratory is not the same as that isolated from the blood culture ✓ Streptococcus pneumoniae will not be recovered after an hour ▪ Isolated organisms must be the same or longer ✓ Must be transported in < 15 minutes Growth of the following… ▪ Enterobacteriaceae PRESERVATION ▪ Streptococcus pneumoniae & Streptococcus pyogenes ▪ Place in incubator or at room temperature ▪ Haemophilus influenzae ▪ Never refrigerate if for bacterial culture ▪ Pseudomonas aeruginosa ▪ Hematology: refrigerate ▪ Neisseria meningitidis ▪ Chemistry and Serology: -20°C ▪ Brucella species ▪ Viral studies ▪ Bacteroides species ✓ refrigerate within 23 hours of collection and kept at ▪ Listeria monocytogenes 4°C up to 3 days ▪ Staphylococcus aureus & Streptococci Gram (-) anaerobes ✓ -70°C if longer delay is anticipated ▪ Candida albicans ▪ Clostridium perfringens CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 PATHOGENS ✓ an opportunistic, atypical fungus infecting ▪ Mycobacterium tuberculosis immunocompromised hosts ▪ Treponema pallidum An alternative: AEROSOL-INDUCED specimen for mycobacterial ▪ Streptococcus pneumoniae and fungal agents ▪ Haemophilus influenzae ✓ Patient is allowed to breathe aerosolized droplets ▪ Streptococcus agalactiae (NEBULIZATION containing 10% 0.85% nacl) until a strong cough ▪ Staphylococcus aureus reflex is initiated ▪ Escherichia coli ▪ Secretions collected thru induction appear watery resembling ▪ Naegleria or Acanthamoeba spp. saliva ▪ Listeria monocytogenes ▪ Contain materials directly from alveolar spaces ▪ Cryptococcus and other fungi ▪ Samples are adequate and acceptable for culture, therefore ▪ Staphylococcus spp. pre-screening is eliminated ▪ Neisseria meningitidis ▪ May obviate doing more invasive procedure such as ▪ Bacteroides spp. bronchoscopy or needle aspiration ▪ Enteroviruses ▪ Instruct patient to bring the sample immediately to the ▪ Staphylococcus epidermidis laboratory ▪ Toxoplasma gondii SPUTUM: ENDOTRACHEAL ASPIRATE APPROPRIATE COLLECTION TECHNIQUES: SPUTUM ▪ Patients with tracheostomies are unable to produce sputum in ▪ Among the least clinically relevant specimens received for normal fashion culture though one of the most numerous and time consuming ▪ Tracheostomy aspirates or Tracheostomy suction specimens are specimens treated as SPUTUM ▪ Not a sample from the post nasal region and is not a spittle or ▪ Lower respiratory secretions are easily collected using Lukens saliva. Comes from deep within the bronchi Trap ▪ Patients are rapidly colonized by gram (-) bacilli and other SPUTUM: EXPECTORATED SAMPLE nosocomial pathogens ▪ No food intake 1-2 hours before collection ▪ Colonization is not clinically relevant but these organisms may be ▪ Brush teeth or gargle with water or saline just before aspirated into the lungs and can cause pneumonia expectoration ▪ Culture results are correlated with clinical signs and symptoms ▪ Practice deep breathing, then cough off sputum secretions ▪ Deep-coughed sample should be expelled directly in sterile TRANSPORT AND STORAGE container ▪ 30 / OIO 0 = No AFB seen in 300 OIF +n = 1-9 AFB/100 OIF C. All Other Specimens (n = actual number of AFB seen, e.g., +4) Gram (-) bacilli = +1, +2, +3, +4 1+ = 10-99 AFB/ 100 OIF Gram (+) cocci arranged in clusters = +1, +2, +3, +4 2+ = 1-10 AFB/OIF in at least 50 fields Pus cells = 35 – 40 / hpf 3+ = more than 10 AFB/ OIF in at least 20 fields Epithelial cells = 2-3 / lpf CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 LESSON 7 ANTIMICROBIAL THERAPY AND SUSCEPTIBILITY TESTING Anti-infective Agents Capable of acting against infection, either by inhibiting the spread of an infectious agent or by killing the infectious agent outright Antibiotics Drugs used to treat bacterial infections Regulates bacterial population or multiplication Differs in chemical composition and on the mechanism of action Chemical substance produced by various species of microorganism with the capacity to kill or inhibit other microorganisms Factors to Consider in Choosing the Most Appropriate Antimicrobial Agents Antimicrobial Drug Therapy Drugs used to treat an infection by killing or inhibiting the growth Identity of the infecting microorganism of the microorganisms that these drugs act primarily against Susceptibility of the microorganism to the antimicrobial agent: Spectrum of Activity Antimicrobial Therapy Action of the antimicrobial agent Use of chemical compounds to treat diseases caused by Patient factors microorganisms Appropriate dosing of the antimicrobial agent Site of Infection Properties of an Ideal Antimicrobial Safety of antimicrobial agent 1. Must have most activity to inhibit/destroy pathogens Cost of the antimicrobial agent 2. With the least toxicity to the host 3. Must be bactericidal rather than bacteriostatic SUSCEPTIBILITY OF THE MICROORGANISM TO THE 4. Must easily penetrate the host cells ANTIMICROBIAL AGENT 5. Should be non allergenic nor should continued Action of the Antimicrobial Agent administration of large doses cause adverse side effects a. Minimum Inhibitory Concentration (MIC) 6. Should be cheap and commercially available To provide effective antimicrobial therapy, the clinically 7. Should be water soluble and stable obtainable antibiotic concentration in body fluids should be greater than the MIC Greatest Sources of Antibiotics the lowest concentration of an agent required to inhibit growth A. Penicillium b. Bacteriostatic B. Streptomyces Chemotherapeutic agent/Antimicrobial agent that inhibits C. Cephalosporium growth and multiplication but not necessarily kill microorganisms D. Micromonospora c. Bactericidal E. Bacillus Chemotherapeutic agent/Antimicrobial agent involved in actual killing of microorganism Spectrum of Activity a. Narrow Spectrum Antimicrobials have limited activity and are primarily only useful against particular species of microorganisms b. Broad Spectrum Antimicrobials are active against both Gram-positive and Gram-negative organisms CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 Site of Infection Basic Principles of Pharmacology a. Adequate levels of the antimicrobial agent must reach the site Pharmacokinetics of infection for the microorganisms be eradicated ← what the BODY does to the b. Route of Administration is determined by: DRUG 1. Properties of the drug PROCESS water or lipid solubility 2. Therapeutic objectives Pharmacodynamics the desire for a rapid onset of action or the need for → what the DRUG does to the long-term administration or restriction to a local site BODY EFFECT Appropriate Dosing of Antimicrobial Agent a. Concentration dependent killing Classification of Antibacterial Agents antimicrobials that show a significant increase in the rate According to Mechanism of Action of bacterial killing as the concentration of antibiotic CLASSIFICATION increases OF ANTIBACTERIAL Drugs that exhibit concentration- dependent killing can AGENTS be given as a once-a-day bolus infusion, which achieves According to According to the high peak levels, favoring rapid killing of the infecting Spectrum of Action Effect on bacteria pathogen Mechanism of Action of Antibacterial Agents b. Time dependent killing ANTIBIOTIC MOA drugs whose killing action is time- dependent Inhibition of protein synthesis bactericidal activity continues as long as serum Inhibition of nucleic acid synthesis concentrations are greater than the Minimum Inhibition of cell wall synthesis Bactericidal Concentration (MBC). Disruption of cell membrane function ✓ lowest concentration of an antibacterial agent Block pathways and inhibit metabolism required to kill a particular bacterium c. Post antibiotic effect persistent suppression of bacterial growth after limited exposure to an antimicrobial agent Patient Factors a. Immune system b. Renal function c. Liver function d. Age e. Blood circulation f. Pregnancy & Lactation Safety of Antimicrobial Agent need to consider the adverse effects brought about antimicrobials Inhibitors of Protein Synthesis need to educate patients regarding the toxicity of AMINOGLYCOSIDES antimicrobials Gentamicin, Amikacin, Tobramycin, Streptomycin, Rifampicin – orange red color of secretions Kanamycin, Netilmicin Clindamycin – diarrhea MACROLIDES Erythromycin, Azithromycin, Clarithromycin Cost of Antimicrobial Agent LINCOSAMIDES need to consider then cost since most patients are not Clindamycin, Lincomycin compliant in finishing their antimicrobial therapy due to its high STREPTOGRAMIN cost Quinupristin-dalfopristin Affordable antimicrobial agents KETOLIDES Generics Act of 1988 (R.A. 6675) Telithromycin OXAZOLIDINONES CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 Linezolid Penicillinase: Stable Penicillins – Cloxacillin, Dicloxacillin, PHENICOLS Methicillin, Nafcillin, Oxacillin Chloramphenicol Amidinopenicillin: Mecillinam TETRACYCLINE Tetracycline, Doxycycline, Minocycline CEPHALOSPORINS GLYCYLGLYCINE First Generation – Cephalexin, Cephalothin, Cefazolin, Tigecycline Cephradine Second Generation – Cefamandole, Cefonizid, Cefuroxime Third Generation – Cefoperazone, Cefixime, Cefotaxime, Ceftazidime, Ceftriaxone, Ceftizoxime Fourth Generation - Cefepime CARBAPENEMS MONOBACTAM Imipenem Aztreonam Meropenem Ertapenem Doripenem BETA-LACTAMASE INHIBITORS Inhibitors of Nucleic Acid Synthesis Some bacteria may produce β-Lactamase in response to NITROIMIDAZOLE β-Lactam drugs; Used in combination with β-Lactam drugs Metronidazole 1. Clavulanic Acid RIFAMYCIN 2. Sulbactam Rifampicin 3. Tazobactam QUINOLONES First Generation (Non-fluorinated) Nalidixic Acid Clinically used combinations: Second Generation (Fluorinated) Ciprofloxacin, Ofloxacin 1. Amoxicillin/Clavulanate Third Generation (Fluorinated) Levofloxacin 2. Ticarcillin/Clavulanate 3. Ampicillin/Sulbactam 4. Piperacillin/Tazobactam Disruption of Cell Membrane Function POLYMYXINS Polymyxin B, Colistin BACITRACIN Block Pathways and Inhibit Metabolism SULFONAMIDES Inhibitors of Cell Wall Synthesis Trimethoprim + Sulfamethoxazole (Cotrimoxazole) CELL WALL SYNTHESIS TRIMETOPRIM BETA LACTAM Trimetoprim Penicillin, Cephalosporin, Carbapenem, Monobactam NITROFURANTOIN GLYCOPEPTIDES Nitrofurantoin Teicoplanin, Vancomycin LIPOPEPTIDES Based on Spectrum of Activity Oritavancin, Telavancin, Daptomycin BETA-LACTAMASE INHIBITORS BETA-LACTAM ANTIBACTERIAL AGENTS Largest group of antibacterial agents PENICILLINS Penicillin: Penicillin Aminopenicillin: Amoxicillin, Ampicillin Ureidopenicillin: Azlocillin, Mezlocillin, Piperacillin Carboxypenicillin: Carbenicillin, Ticarcillin CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 Effect on Bacteria MECHANISMS OF BACTERIAL RESISTANCE I. Intrinsic Resistance Related to structural features and are usually determined by chromosomal genes II. Mutational Resistance Results from a chromosomal mutation that renders the bacterium unable to interact with the antibacterial III. Acquisition of Resistant genes Is most often plasmid-mediated MICROBIAL RESISTANCE DRUG FAST According to WHO... A term denoting the resistance of a pathogen to Worldwide, estimated half of all medicines are inappropriately chemotherapeutic agents prescribed, dispensed or sold, and half of all patients fail to take their medicine properly MULTIDRUG RESISTANT ORGANISMS (MDRO) Two-thirds of global antibiotic sales occur without any microorganisms (predominantly bacteria) resistant to one or more prescription classes of antimicrobial agents 90% of injections are estimated to be unnecessary MULTIDRUG RESISTANT (MDR) MICROBIOLOGY LABORATORY ROLE non-susceptibility to at least one agent in three or more Right specimen for right results... antimicrobial categories Important to: optimize chance of microbiological diagnosis and help tailor therapy to specific pathogen EXTENSIVELY DRUG RESISTANT (XDR) non-susceptibility to at least one agent in all but two or fewer Should be: antimicrobial categories (i.e. bacterial isolates remain susceptible reliable & adequate to only one or two categories) appropriately transported and processed Obtain appropriate cultures before initiating antibiotic therapy PAN DRUG RESISTANT BUT this should not delay administration of antibiotic non-susceptibility to all agents in all antimicrobial categories (i.e. Proper coordination is the KEY! no agents tested as susceptible for that organism) YOU ARE the Medical Technologist on duty…what to do!? IS AN ANTIBIOTIC INDICATED? Determining the TRUE PATHOGEN.... Seek the RIGHT advice… Right specimen for right results… Based On: Determining the TRUE PATHOGEN.... Appropriate dose of the patient age appropriate drug.... Any special host factors? Best route of diagnosis and site of infection administration? Appropriate dose given? epidemiological data hospital-acquired vs. community-acquired Susceptibility Testing prior antibiotic use Factors to consider when determining whether SENSITIVITY culture data TESTING is warranted: Defined as microorganisms that are not inhibited by usually 1. Body site where the organism was isolated achievable systemic concentration of an antimicrobial agent with 2. Testing of normal flora or of possible contaminant is normal dosage schedule and/or fall in the minimum inhibitory avoided concentration (MIC) range 3. Presence of other bacteria and the quality of specimen is scrutinized – Only pure cultures with no contaminants BACTERIAL RESISTANCE ACQUISITION and of significant numbers of CFU (10 CFU/ ml. should 1. Enzymatic inactivation of antibacterial agents be tested 2. Modification of cell wall integrity 4. Host’s status is considered 3. Alteration of target molecules 4. Development of alternate pathways Selecting antimicrobial agents for testing should be formulated by: 5. Active exclusion of the antimicrobial agent from the a. Laboratory personnel bacteria b. Infectious disease department 6. Development of tolerance c. Pharmacy CLINICAL BACTERIOLOGY 1ST SEM - MIDTERMS Microbiology and Parasitology (20%) Hannah Fierela B. Limpiado S/Y 2022-2023 BSMT-L3 METHODS OF SENSITIVITY TESTING I. DILUTION METHODS Minimal Inhibitory Concentration (MIC) is determined MIC is the lowest concentration of an agent required to inhibit growth A. Broth Macrodilution (Tube Dilution) Tests Uses tubes with a serial dilution of antimicrobial The last tube (lowest concentration) which shows no growth determines the MIC B. Broth Microdilution Tests Same principle as macrodilution but uses microtiter plates instead of test tubes C. Agar Dilution Tests Series of plates containing various concentration of antimicrobial agent is inoculated with standard number of test bacteria using a multi-pronged replicating device. (Steer’s replicator) II. DISK DIFFUSION TESTING/KIRBY- BAUER TEST For rapid growing aerobes Highly standardized method for determining the susceptibility or resistance of bacteria to antimicrobial agent; Standardized inoculum is streaked in Mueller-Hinton Agar using overlap streak method. Zones of inhibition is measured after overnight incubation and reported as SENSITIVE, INTERMEDIATE or RESISTANT. Modified methods can be used for testing slow-growing or fastidious bacteria since MHA cannot support the growth of all bacteria Haemophilus – Haemophilus Test Medium (HTM) Streptococcus – MHA with 5% sheep’s blood Neisseria – GC Agar base with supplement Anaerobes – Wilken’s-Chalgren Agar/Wilkins-Chalgren Broth METHODS OF SENSITIVITY TESTING III. AUTOMATED METHODS IV. NEWER NON-AUTOMATED METHODS A. Alamar System B. E-Test / Epsilometer Test C. Spiral Gradient Endpoint System E-Test MIC of the Bacteria can be read Directly Kirby Bauer Disk Diffusion Method