Microbiology Course: Diagnosis of Infectious Diseases Lecture PDF

M ICROBIOLOGY Course Diagnosis of Infectious Diseases CPC C L O W N I N P L U S C R O W N  Moayad Odat Introduction Organization of pathology department (Labs) :...

M ICROBIOLOGY Course Diagnosis of Infectious Diseases CPC C L O W N I N P L U S C R O W N  Moayad Odat Introduction Organization of pathology department (Labs) : Clinical Microbiology Lab (CML) : Mission : active participation in management (Dx & Rx) of patients with infectious diseases. Approach a patient with infectious disease: Patient History : document symptoms Physical Examination: identify clinical signs. Differential Diagnosis: list potential diagnoses Investigations : tests to refine the diagnosis (laboratory & radiological) Final Diagnosis : confirm the diagnosis Treatment plan : outline appropriate antimicrobial therapy. Four major day-to-day responsibilities : 1- Processing of Clinical Specimens. 2- Isolation of potential Pathogens. 3- Identification of isolated Pathogens. 4- Antimicrobial susceptibility testing (AST) Other responsibilities : Hospital Outbreak (more than one case of the same MOs in the hospital) Recognition, notification & source identification : — Environmental samples processing — Health care providers screening. Organization of CML (Sections) : Bacteriology Mycobacteriology (TB) : — Only in large hospitals & medical centers (part of the CML) — In small hospitals : specimens sent to a reference lab Virology : — Only in large hospitals & medical centers (part of the CML) — In small hospitals : specimens sent to a reference lab Immunology : — Large hospitals & medical centers : clinical immunology lab (independent of CML) — Small hospital : part of CML Mycology Parasitology 1  Moayad Odat Clinical Specimens (Samples) Collected from patients & used for diagnosis or follow progress of IDs. Three components for high-quality specimens : 1- Selection : Appropriate specimen type Blood sample for toxin mediated infections (cholera ,botulism ,diptheria & scarlet fever) will be rejected 2- Collection : Clear detailed instructions to patients Before antimicrobial treatment Sufficient quantity Use appropriate container : — Sterile : urine ,CSF & blood samples — Clean : stool sample Properly labeled (patient name & number) 3- Transport : Sent immediately (ASAP) The specimen should be appropriately stored in the cases of expected delay Adequately completed request forms (not completed → rejected) NOT transported properly: — Potential pathogen destroyed (not detected) — Masked by overgrowth of flora — MisIded by contaminants. Rejection criteria (No lab processing) : Unlabeled or mislabeled sample Leaked containers Quantity Not Sufficient (QNS) The sample received in formalin (should be in saline) Specimen processing : Vary according to specimen type. Systematic processing steps : 1- Macroscopic examination (naked eye) 2- Microscopic examination (wet mount or/& staining) 3- Inoculation of appropriate culture media : — Pure significant growth of expected pathogens — Identification & AST. Direct Staining : Applied on specimen directly Observing: MOs ,inflammatory cells & others (epithelial cells & protein debris) Indirect Staining: Applied on isolated colonies (on next days after cultivation) Observation of MOs ONLY 2  Moayad Odat Notes: Vesicle fluid or scraping → ID of viral infections. Hair, Nail clippings & Skin scraping → ID of fungal infections. Skin snip, Scotch tape preparation → ID of parasitic infections : Skin snip : Trichinella spiralis Scotch tape preparation : Enterobius vermicularis Urine Specimens The most common clinical specimen sent to the CML Urine is ordinarily Sterile in the urinary bladder. Collection for culture : 1- Clean-Catch Mid-Stream (CCMS) : Clean-Catch : washing area around external meatus of urethra with soap & water to remove skin flora Mid-Stream : discarding initial portion of urine stream to flush MOs in distal urethra Used with persons who able to follow instructions 2- Catheterized urine : Intermittent catheterization (Superior for Testing) : neurological bladder In-Situ catheterization (Colonization Risk) : bedridden or paralyzed patients 3- Urine bag : infants and young children (not toilet-trained) 4- Suprapubic needle aspiration (SPA) : The most sterile urine sample by bypassing the urethra. The Gold standard method for urine sampling Invasion method Urine processing : Within 30 min of collection (24 hr if refrigerated at 4°C) Calibrated loops : Metallic wire or disposable plastic Specific volumes of urine : — 0.01 mL (dilution factor = 100) — 0.001 mL (dilution factor = 1000) Inoculation of solid media : Blood agar MacConkey agar Incubation at 37°C overnight. Quantitative bacterial colony count : CFU (colony-forming units) : — Number of viable bacteria / mL urine — Number of colony X dilution factor (100 or 1000). Note : Urine specimen is the only quantifying sample 3  Moayad Odat Results & Interpretation : Criteria determining clinical significance : Patient signs & symptoms & sex (females have more risk of UTIs (short kinked urethra)) Patient age : children are more susceptible to get UTI Patient history and location (Urology, ED ,ICU) Pyuria (high PMNLs in urine) : clinical significance (indicates UTI) Specimen type. Pure growth : one MO (pathogen) || ≥2 different MOs (contaminants) Significant Count || Insignificant Growth (ISG) : Tube 2 > Tube 3) Clear supernatant || Possible clot — Hemorrhagic blood: Constant blood quantity in tubes (Tube 1 = Tube 2 = Tube 3) Xanthochromic supernatant || NO clot Xanthochromic (yellowish) : Jaundice 2- Centrifugation : Sediment : — For microscopic examination — Staining: Gram stain & Methylene blue — Preliminary (Interim) report by Telephone — Culture: Media (blood ,MacConkey ,chocolate & SDA agars) — Colonies: Identification & AST. Supernatant : — Slide latex agglutination test — To detect bacterial capsular Ags — In pre-treated patients 3- Chemistry : ↓ Glucose & ↑ Total Protein → bacterial infection 4- Hematology : Bloody sample → will Not be counted WBCs → Count → Pleocytosis → Different predominance : — ↑ PMNLs (neutrophils) : bacterial infection — ↑ Lymphocytes : viral infection RBCs 5- Final report : negative ONLY after 48 hrs of incubation. Microbial Pathogens in meningitis : Leading major bacteria : H.influenza, meningococcus & pneumococcus. Neonates : Group B Streptococcus (Streptococcus agalactiae) GN bacilli: E. coli ,Enterobacteriaceae ,Klebsiella & Salmonella Listeria monocytogenes. VP shunt : CONS (S. epidermidis), Acinetobacter spp, Pseudomonas aeruginosa & S. aureus. 7  Moayad Odat Immune-compromised : Listeria in adults & Cryptococcus neoformans in all age groups. Fungus: Cryptococcus neoformans. Protozoa : Free-living amebae: Naegleria & (Acanthamoeba spp & Balamuthia mandrillaris) Other protozoa: Toxoplasma, & Trypanosoma. Nematodes: Angiostrongylus cantonensis. Aseptic meningitis : CSF negative culture High cells number (less serious than common bacterial meningitis) Short duration of symptoms → most likely viruses If the child was unwell more than a week → Mycobacterium Tuberculosis Wound Specimens Preferably ASPIRATE / PUS (collected by small needle & syringe) Swab : Liable for frequent contamination with normal flora Often dry out before processing Swabs for MTB, & fungi (Except Candida) will be Rejected Adequately completed request form (indicate TYPE of wound Infection) : Burn → Pseudomonas Postsurgical → Staph Dog bite → Pasteurella multocida Gonococcus culture Gonococcus: Fastidious || Microaerophilic || Capnophilic MOs GN cocci || Oxidase test +ve. Urethral discharge in Lab (superior & preferable) Alternatives : urethral, vaginal, cervical, throat, & rectal swabs Use ONLY : dacron, calcium alginate, or nontoxic cotton swabs. Avoid ordinary cotton swabs (toxic FAs). Never refrigerate Gonococcal swabs (low temp is lethal). Sample processing : Stains : Gram stain → GN intracellular bean-shaped diplococci (DDx : Veillonella spp) Methylene blue stain → Cellular details Inoculation (immediate inoculation) : Chocolate agar New York City agar Martin-Lewis or Thayer-Martin agars Inoculation in CO2 (Capnophilis) In the cases of expected delay : transport culture medium with 5-10% CO2 Incubated at 37°C overnight. 8  Moayad Odat Throat Swabs Routinely for Group A streptococci (S.pyogenes) : Gram positive cocci (beta hemolytic) Bacitracin sensitive Lancefield grouping by slide agglutination test Specific (upon request): Corynebacterium diphtheriae Gonococcus (Neisseria gonorrhoeae) Sputum Sample Pus accumulating deep within lungs in lower RTIs (pneumonia & TB) Collection : Ambulatory alert patients : expectorated (mouth hygiene / coughing) ICU patients : induced (Respiratory staff : Endotracheal or Tracheal Aspirates) Quality : Sputum : ≥25 pus cells (neutrophils) / HPF Saliva : ≥25 epithelial cells / HPF Microbial Pathogens : AIDS patients: Pneumocystis jirovecii (carinii) pneumonia (fungus NOT protozoan) Community: Pneumococcus, H. influenzal. Cystic Fibrosis : Pseudomonas aeruginosa ICU : MDR (Acinetobacter, MRSA) Feces (Stool) specimen Stool (superior & preferable) vs Rectal swab (alternative). Container : clean (Not necessarily sterile) Transport & delivery to Lab : Add preservative to maintain pH 7.0 Delay → ↓ Temp → ↓ pH → death of Salmonella & Shigella Processing: immediate / ASAP (< 2 hrs) Media preparation : NO autoclaving of media Inoculation : First : enrichment broths (Selenite-Fecal or Tetrathionate) Second (sub culture) onto selective media : Salmonella Shigella Agar (SSA) ,Deoxycholate Citrate Agar (DCA). Microbial Pathogens : Routine (Salmonella & Shigella) Cold climate (Campylobacter & Yersinia) ≤1 year old infant (E. coli O157-H7) Outbreak (Vibrio cholerae) Colon is anaerobic : Clostridium difficile (toxin detection) Clostridium perfringens (food poisoning). Stool analysis for Ova Parasite : Intestinal protozoa (Entamoeba, Giardia, Balantidium, Cystoisospora, Cyclospora, Cryptosporidium), & Helminths. 9