2024 Clinical Microbiology Handout-1.pdf

Clinical Microbiology Courtesy of Sandra Comand Clinical Manager Brant Community Healthcare System Specimen Process SPECIMEN IS COLLECTED SPECIMEN IS PROCESSED IN LABORATORY LABORATORY ISSUES A REPORT Importance of Correct Specimen Collection A sufficient quantity of specimen must be collected Speci...

Clinical Microbiology Courtesy of Sandra Comand Clinical Manager Brant Community Healthcare System Specimen Process SPECIMEN IS COLLECTED SPECIMEN IS PROCESSED IN LABORATORY LABORATORY ISSUES A REPORT Importance of Correct Specimen Collection A sufficient quantity of specimen must be collected Specimen should be representative of the infectious process – Sputum, not saliva – Swab from the depth of the wound, not from its surface Care must be taken to prevent contamination of the specimen by using only sterile equipment and aseptic precautions Meaningful specimens must be collected before the administration of antimicrobials (e.g. CSF, blood cultures, tissues) Specimen must be taken to the laboratory and examined promptly – Importance of storing at appropriate temperatures Specimen Collection: Containers MICROBIOLOGY SPECIMEN CONTAINERS Pediatric Bottle 2-4ml Amies Transport Medium superficial wounds catheter sites vag / cerv throat donor bones surveillance Aerobic Bottle 8-10ml P.R.A.S. Transport Anaerobe investigation OR surgical specimens OR tissues (small) body fluids aspirates Anaerobic Bottle 8-10ml Enteric Transport faeces culture Sterile Container urine tissue (large) bone chips C.difficile body fluids Ova & Parasites parasitology Laboratory Specimen Process Day 1: DIRECT STAINS / CULTURE PLATES Day 2,3 etc.: INCUBATION / EXAMINATION PATHOGENS IDENTIFIED / SENSITIVITIES REPORT ISSUED Microscopy Bright‐field (light) ‐ visible light is passed through the specimen, a series of lenses that reflect the light – Stain technique: Gram stain – Target: Bacteria / Fungi Classifications Bacteria Mycoplasma Virus Chlamydia Fungus 0.5 µm 0.1 µm 20 – 30 nm 0.25 – 0.35µm 2 – 60 µm Prokaryotic Prokaryotic Intracellular Intracellular Eukaryotic Cell wall No cell wall No cell wall Cell wall DNA + RNA DNA DNA or RNA DNA + RNA Cell wall (chitin within cell wall) Binary Grows on Particles nonliving media replicate within host cell Binary Does not grow on nonliving media Reproduction via sexual or asexual spores Prokaryotic: absence of a nuclear membrane, divides by binary fission, includes bacteria + blue green algae Eukaryotic: cells have a nuclear membrane, divides by mitosis, includes humans, plants, animals, fungi, and algae Day 1: DIRECT STAINS / CULTURE PLATES Lab Process: Gram Stain – Gram Positive Gram Positive cells resist decolourization of the CVI complex (crystal violet/iodine) = purple Cocci in pairs / chains (Strep) Diplococci (Strep. pneumoniae) Bacilli (Clostridia, diphtheroids) Cocci in clusters (Staph) Lab Process: Gram Stain - Gram Negative Gram negative cells decolourize rapidly with the acetone/alcohol and then hold on to counter stain of safranin = pink Bacilli (Coliforms) Diplococci (N. meningitidis, N. gonorrhea) Coccobacilli (Haemophilus) Lab Process: Day 2,3 etc.: INCUBATION / EXAMINATION PATHOGENS IDENTIFIED / SENSITIVITIES Lab Process: Bacteria Identification Gram stain morphology Colour – positive vs. negative Shape – cocci, bacilli Growth requirements / Incubation Aerobic, facultative anaerobe, strict anaerobe, capnophilic (fastidious) Plate morphology Blood, selective media Biochemical tests DNA testing Lab Process: Gram Positive Cocci Micrococcaceae Staphylococcus aureus Staphylococcus epidermidis Coagulase Negative Staphylococcus Streptococcaceae Streptococcus pyogenes (Group A Strep) Streptococcus agalaciate (Group B Strep) Streptococcus pneumoniae Viridans Group Streptococcus Enterococcus faecalis Enterococcus faecium Lab Process: Staphylococcus aureus Lab Process: Gram Negative Cocci Aerobic Neisseria gonorrhoeae Neisseria meningitidis Moraxella catarrhalis Anaerobic Veillonella Lab Process: Neisseria gonorrhoeae Lab Process: Gram Positive Bacilli Facultative Anaerobic, Spore formation Spore Positive –Bacillus species Bacillus anthracis (anthrax) Bacillus cereus Spore Negative –Coryneforms (diphtheroids) Corynebacterium diphtheriae (diphtheria) –Listeria group (Listeria monocytogenes) Anaerobic, Spore formation Positive –Clostridium species Clostridium botulinum (botulism) Clostridium difficile Closterium perfringens (gangrene) Clostridium tetani (tetanus) Negative –Propionibacterium acnes (acne) –Actinomyces Lab Process: Spore stain Lab Process: Clostridium difficile Pseudomembranous colitis Antimicrobial associated colitis occurs after prolonged use of broad‐spectrum antimicrobials Toxin A / Toxin B (enterotoxins released) Lab Process: Clostridium difficile Lab Process: Gram Negative Bacilli – Facultative Anaerobic Enterobacteriaceae Lactose fermenters –Escherichia coli (E. Coli) –Escherichia coli 0157:H7 –Klebsiella pneumoniae –Enterobacter Non lactose fermenters –Proteus –Providencia –Morganella –Enteric pathogens: Salmonella, Shigella, Yersinia Others Vibrio, Aeromonas, Plesiomonas Lab Process: Escherichia coli Lab Process: Gram Negative Bacilli – Microaerophilic / Anaerobic Microaerophilic Campylobacter jejuni Helicobacter pylori Anaerobic Bacteroides, Porphymonas, Prevotella, and Fusiforms Lab Process: Gram Negative Bacilli Miscellaneous Fermenters, Non fermenters Oxidase Motility Growth requirements Anaerobic MacConkey agar Examples: Acinetobacter, Pasteurella (animal bites), Pseudomonas Non fermenter Oxidase – Positive: Pseudomonas aeruginosa – Negative: Stenotrophomonas maltophilia Lab Process: Pseudomonas aeruginosa Susceptibilities MIC – minimum inhibitory concentration Different dilutions of the antibiotic are evenly distributed throughout a panel in which the target organism is added Growth in well = resistant No growth in well = sensitive The first well that show no growth would be the MIC Breakpoint Paper disks impregnated with the antibiotic are placed on agar that is inoculated with target organism Growth up to disk = resistant No growth in a zone around disk = sensitive MYCOPLASMA: Mycoplasma hominis NO cell wall Found in: Urogenital tract (prostatitis, vaginosis, pelvic inflammatory disease (PID), neonatal infections) Respiratory tract Rx: Tetracyclines, Fluroquinolones, Clindamycin CHLAMYDIA: Chlamydia trachomatis Obligate intracellular parasite Detections: – NAAT for detection (preferred) – Use fluorescent microscope to see Presentations: – Non gonococcal urethritis (NGU), cervicitis, PID, conjunctivitis, trachoma Rx – Azithromycin, Doxycycline FUNGUS: Aspergillus fumigatus Fungus is much larger Dichotomous branching ~ 45 Found in sputum, leading to: – Pulmonary aspergillosis – Systemic dissemination Opportunistic invader in immunodeficient individuals Rx: – Flucytosine or Amphotericin B YEAST: Candida albicans Most common type of yeast Pseudo hyphae – chain of budding yeast Normal flora of mucous membranes in the respiratory, gastrointestinal, and female genital tracts Predisposing factors to infections are : Immunocompromised Diabetes Administration of antimicrobials Presents as: Thrush Vaginitis Nail infection Skin infection Rx – antifungals (‘azoles’) PARASITE: Malaria falciparum Parasitic infection transmitted by female Anopheles mosquitoes CBC smear required Fevers in returning travelers Prevention: – Chloroquine and hydroxychloroquine, Malarone, Doxycycline, Larium/Mefloquine ‐ SE https://www.canada.ca/en/public‐health/services/catmat/canadian‐ recommendations‐prevention‐treatment‐malaria.html Laboratory: Issuing a Report Normal flora Always there (nonsterile sites) Flora is specific to the body site (skin, respiratory, etc.) Colonizer Non‐invasive and no host response Pathogen Frank vs. potential pathogen Invasive and host response Body site Contaminant Environmental, blood cultures, sputum Laboratory: Possible Reports Bacteria present in the Gram stain, no growth on culture, possible reasons: Urine Report Case #1: Colony count: 10 – 100 x106 org/L Culture: Mixed growth of doubtful clinical significance Urine Report Case #2: Colony count: >100 x106 org/l Culture: Escherichia coli Sensitivity: Ampicillin, Cefazolin, Ciprofloxacin, and SMX/TMP = Sensitive Laboratory: Possible Reports Sputum Report Case: Gram stain Many Polymorphs seen Moderate Gram‐positive cocci in clusters seen Many Gram‐positive diplococci seen Culture Moderate growth of Respiratory flora Heavy growth of Streptococcus pneumoniae Sensitivity: Erythromycin, Levofloxacin, Penicillin = Sensitive Antimicrobial Resistant Organisms (AROs) Identified with DNA and PCR testing MRSA – Methicillin Resistant Staphylococcus aureus (nares/rectal) –Resistant to beta lactam antibiotics plus other drugs VRE – Vancomycin Resistant Enterococci (rectal) ESBL – Extended Spectrum Beta lactamase (rectal) MDRPA – Multi‐Drug Resistant Pseudomonas aeruginosa (rectal) CPE – Carbapenemase Producing Enterobacteriaceae (rectal) AROs Key Points Surveillance –Determines baseline of activity within the organization Isolate / Cohort patients –Prevents transmission from occurring Contact Precautions –PPE!! Signage –Such as contact / isolation precaution requirements Isolation carts Fact sheets (patients / families) Coronavirus (SARS-CoV-2 or COVID-19) Coronavirus first identified 1980s Similar but not the same as: – SARS – MERS Virus – enveloped, positive ssRNA Respiratory droplet and airborne transmission Variants of Concern (currently!) – B.1.1.529 – OMICRON (with BA.2 / BA.5 / BA.7 / XBB.1.5 or Kraken / EG.5) – Subvariant of Omicron – B.2.86 known as Pirola (including JN.1) circulating now Previous variants of concern – UK: B1.1.7 ‐ ALPHA – SA: B.1.351 ‐ BETA – Brazil: P.1 ‐ GAMMA – India: B.1.617.2 – DELTA More detail will be discussed in the Respiratory Lecture later in the term Influenza Type A or B Pandemic – H1N1 in 1918 Orthomyxoviridae family – A, B, C, D = subtypes/strains Virus – enveloped, negative ssRNA Protein spikes to determine A subtype – HA – hemagglutinin – 13 major types (18 possible) – NA – neuraminidase – 9 major types (11 possible) Respiratory droplet and airborne transmission 2023: H1N1 & H3N2 circulating RSV (Respiratory Syncytial Virus) Paramyxoviridae family Virus – enveloped, negative ssRNA Respiratory droplet, airborne transmission, direct contact 2 major subtypes – A and B Biggest concern for young children – Bronchiolitis – Pneumonia Infection Control Resources Local Public Health Units – List of communicable disease and levels for notifications Public Health Agency of Canada – Disease Prevention & Control Guidelines – https://www.canada.ca/en/public‐health/services/reports‐publications/disease‐prevention‐control‐ guidelines.html IPAC – Infection Prevention and Control Canada – https://ipac‐canada.org/ Provincial Infectious Disease Advisory Committee on Infection Prevention and Control (PIDAC‐IPC) – https://www.publichealthontario.ca/en/about/our‐organization/external‐advisory‐committees/pidac‐ ipchttp://www.health.gov.on.ca/english/providers/program/infectious/pidac/pidac_mn.html Association for Professional in Infection Control & Epidemiology – https://apic.org/ Prevention of Healthcare Associated Infections Sandra Callery RN MHSc CIC