Lecture 7 PDF - Diagnosing Infectious Diseases

Diagnosing of Infectious Diseases  Organization of the Pathology Department Anatomical Pathology Clinical Pathology Electron Microscopy Clinical Microbiology Morgue (Autopsy) Clinica...

Diagnosing of Infectious Diseases  Organization of the Pathology Department Anatomical Pathology Clinical Pathology Electron Microscopy Clinical Microbiology Morgue (Autopsy) Clinical Immunology Histopathology Hematology/Hematopathology Cytology Lab Blood Transfusion (Bank) Cytogenetics Lab Clinical Chemistry  Organizations of CML:  Bacteriology  Mycobacteriology  part of CML in large hospitals & medical centers BUT in smaller hospitals specimens are sent to reference lab  Virology  the same as mycobacteriology  Immunology  independent from CML in large hospitals BUT a part of CML in smaller hospitals  Mycology  Parasitology Clinical microbiology lab  The main mission of CML: active participation in management of patients with infectious disease.  Four major day-to-day responsibilities: 1. Processing of Clinical Specimens 2. Isolation of potential Pathogens 3. Identification of isolated Pathogens 4. AST (antibiotic sensitivity test)  Other responsibilities include:  Hospital Outbreak Recognition  Notification & Source identification (Environmental Samples Processing & HCPs Screening) Clinical Specimens (Samples)  collected from patients & used for diagnosis or follow progress of infectious diseases.  3 components for High-quality specimens: 1. Selection. (what is the appropriate sample to be selected?) 2. Collection:  Instruction for best collection of specimens: o detailed instructions to patient o standard precautions o Pre antimicrobial therapy (must) o sufficient quantity o sterile container (stool?) o properly labeled 1  3. Transport:  As soon as possible (no delay)  Storage  Adequately completed request forms  If not transported appropriately; o potential pathogen destroyed (NOT found) o masked by overgrowth of flora & misled by contaminants  You can reject the specimen (NO Processing) when; o Un / mis-labeled container o leaked, QNS (quantity not sufficient) o sent in formalin  If accepted  Lab Processing Processing Clinical Specimens  Vary according to specimen Type  Systematic processing steps 1. Macroscopic examination by Naked-eye 2. Microscopic examination by wet mount or staining  Staining: o Direct: on specimens; what could be seen?  MOs+ inflammatory cells+ other (epithelial cells & protein debris) o Indirect: on next day’s isolated colonies (MOs ONLY). 3. Inoculation of appropriate culture media pure growth 4. Identification & AST Specimen & Pathogens isolated o Direct: on specimens from sterile sites Throat swabs Bacteria, Viruses o Indirect: on next day’s isolated colonies Hair ,Nail clippings, Skin scrapings  Fungi Skin snip, Scotch tape preparation Parasite Vesicle fluid or scraping Viruses Urine  Ordinarily Sterile in urinary bladder  Collection of specimen: 1. Clean catch mid-stream urine in sterile container: (aware patient) o Clean-Catch (washing area around external urethra with soap & water to remove skin flora) o Mid-Stream (discarding initial portion of urine stream to flush MOs in distal urethra) 2. Catheterized urine (bedridden patients) 3. Urine bag (babies) 4. Suprapubic needle aspiration (SPA): Gold standard &Invasive  Rejection: urine specimen in leaked container.  Urine Processing: o Timing:  within 30min of collection  within 24hr if refrigerated at 4°C 2  o Calibrated loops:  Metallic wire (not used currently) or disposable plastic  Multiple sizes, each carries specific volumes of urine - 0.01 mL - 0.001 mL  Inoculation of culture media (Blood Agar & Macconky agar)  Incubation at 37°C overnight o Quantitative bacterial colony count:  CFU (colony-forming units)  number of viable bacteria per mL urine  CFU = colony number x dilution factor  Diluting factor: - 0.01 calibrated loop diluting factor= 1000 CFU/ml - 0.001 calibrated loop diluting factor= 100 CFU/ml  Results & Interpretation: o Criteria determining clinical significance:  Patient: age, sex, history, location  Pyuria: high PMNLs (infection)  Specimen type  Growth: pure; 1 MO vs ≥2 different MOs (contaminants)  Count & identity of pathogen o Pure significant counts indicating UTI:  CCMS 100,1000 (1X105 ) CFU/mL  Catheter urine 1000 (1X103 )  SPA any growth is significant o Pyuria: (≥10 WBCs/ cmm)  Sterile Pyuria: pyuria without bacteriuria - Symptomatic: hot metallic loop, pre-antimicrobial therapy, atypical/ fastidious MOs - Not symptomatic: pregnancy, vaginal discharge, stones, DM, interstitial nephritis  Bacteriuria without pyuria is usually NOT significant  Urinary tests (very important) o Leukocyte esterase test (indicates pyuria):  LE enzyme is produced by most granulocytic WBCs (PMNLs, eosinophils, basophils) & monocytes o Nitrite test (nitrituria) (indicates bacteriuria):  detects gram (-) MOs able to reduce nitrate to nitrite  not detected: Staph saprophyticus, enterococcus, P. aeruginosa, Acinetobacter, Adenovirus) Blood  Blood is usually sterile  Any isolate from blood should always be considered significant until proven otherwise 3  Toxin mediated infections: ‫مهم جدا‬ - Cholera  Indications of blood culture - Diphtheria o Diagnosis of most bacterial & fungal SYSTEMIC Infections: - Bordetella pertussis 1. Bacteremia (bacteria in PBS) in: - Bacterial meningitis, endocarditis, pneumococcal pneumonia, tularemia - Typhoid fever, Salmonella Infections, UTIs, brucellosis, plague, anthrax - wound Infections caused by -hemolytic strep, staph & fungi - temporary bacteremia may occur following oral surgery, tooth extraction or even aggressive tooth brushing 2. GNB Septicemia (bacteria & their toxins in PBS). 3. IV lines  Blood Culture Specimens o Aseptic technique: gloves, skin disinfection (alcohol, iodine) to prevent contamination with skin flora. o 1 aerobic, & 1 anaerobic blood culture bottles from refrigerator to room temp. o Remove foil, disinfect rubber tops & inject volume specified. o Transport to Lab ASAP & Incubate at 37°C. o NEVER refrigerate  Conventional Processing o Daily morning inspection of undisturbed blood culture bottle for indicators of microbial growth:  turbidity, gas production, hemolysis or white colonies on sedimented blood cells layer - Present→ Gram stain, inoculation of solid media (Blood, Chocolate, MacConkey, SDA) →isolation, Identification, & AST - Absent→ negative result after 5-7 days of Routine incubation o Prolonged incubation:  Endocarditis & fungi (3 wks)  Brucella (6-8 wks)  Automated B/C Systems o Incubator & continuous detector of O2 consumption & CO2 production by microbial metabolism. 1. BACTEC  uses internal fluorescence sensor 2. BacT/ALERT 3D uses internal colorimetric sensor 3. Versa TREK uses external pressure sensor w measures pressure changes in bottle headspace  Contamination: o Pseudobacteremia: false positive detection of microbial growth. (neonates) o Ward-based: - During specimen collection - Skin flora of patients (3%): CONS, diphtheroids & propionibacterium, growth within 1-3 days o Lab-based: after routine Sub-culturing, same MO from different patients at different locations 4   Troubleshooting Gram stain Sub- culture Microbe GPC No Growth anaerobic staph (peptococci), anaerobic strep (peptostreptococci) or pyridoxine-dependent nutritionally variant streptococci (Abiotrophia) GPC GNC Acinetobacter GNC No Growth Anaerobic (Veillonella sp). GPB No Growth Anaerobic (Propionibacterium sp). GNB No Growth HACEK fastidious GN bacteria, oropharyngeal normal flora, culture- negative endocarditis, native valves, most common GNB cause of endocarditis among people who do not use IV drugs. Cerebrospinal Fluid (CSF) HACEK: - H: Hemophilus spp  When the ICP is increased Lumber Puncture is contraindicated  - A: Actinobacillus because of coning (herniation through foramen magnum) actinomycetemcomitan  Collection: - C: Cardiobacterium hominis o by physician, by LP (spinal tap), under strict surgical asepsis - E: Eikenella corrodens o 3 labelled sterile tubes: (1) chemistry, (2) CML, & (3) hematology. - K: Kingella spp  Transport & delivery: ASAP, NO delay, NO refrigeration.  Lab Processing (emergency): immediate upon receipt/ without any delay.  CSF Processing: o Macroscopic Examination:  Crystal clear  normal  Cloudy or turbid ↑[protein or lipid], presence of WBCs.  Bloody: - trauma (blood increase with time, clear supernatant, possible clot) - hemorrhage (constant, xanthochromic supernatant, NO clot).  Xanthochromic (yellowish) jaundice o Centrifugation  Sediment: 1. Microscopic examination Stains → Preliminary report by Telephone 2. Culture Media (Blood, Chocolate, MacConkey, SDA) 3. Identification & AST  Supernatant Slide latex agglutination test (bacterial capsular Ags, pre-Rx) o Molecular  Chemistry ( Glucose &  Total Protein)  Hematology: WBCs & RBCs. o Final report: negative ONLY after 48hrs of incubation. 5   Microbial Pathogens o Leading major bacteria: Hflu, meningococcus, & pneumococcus. o Neonates: GBS, GNB (mainly E. coli K1, & other members of Enterobacteriaceae as Klebsiella spp, awa Salmonella in infants few months old in some countries with warm climate) & Listeria monocytogenes. o VP shunt: CONS (S. epidermidis), Acinetobacter spp, Pseudomonas aeruginosa, other GNB, S. aureus. o IS↓: Listeria in adults, & Cryptococcus neoformans in all age groups. o Fungus: Cryptococcus neoformans. o Free-living amebae: Naegleria, & (Acanthamoeba spp & Balamuthia mandrillaris). o Other protozoa: Toxoplasma, & Trypanosoma. o Nematodes: Angiostrongylus cantonensis. o Aseptic meningitis: CSFNegative culture & cells number, less serious: - short D symptoms → most likely viruses - child unwell >1wk → MTB. Wound Specimens  Collection: o Preferably ASPIRATE (syringe) o Not Swab contamination with normal flora & dry out before processing  Rejection: swabs for MTB, & fungi (Except Candida).  Request form (adequately completed): always indicate TYPE of wound Infection: burn, postsurgical, or dog bite (Pasteurella multocida) GC Cultures  Urethral discharge in Lab is preferable vs urethral swabs  Use ONLY Dacron, calcium alginate, or nontoxic cotton swabs.  Avoid ordinary cotton swabs (toxic FAs).  Never refrigerate GC swabs (low temp is lethal). RS samples  Throat swab: o routinely check for strep. Pyogenes (group A, β-hemolytic) - Hemolysis, Bacitracin S testing, Lancefield grouping by slide latex agglutination test o others: gonococcus, Corynebacterium diphtheriae  Sputum: worst yield o Microbial Pathogens - Pneumococcus, H.flu - MDR in ICU patients: Acinetobacter, MRSA - Colonization: Staph aureus, GNB, Candida, 6 Feces (Stool)  Stool is more preferable than rectal swab  Collection: clean (unsterile) container with tight lid, NOT contaminated  Add preservative to maintain pH7.0 as delay→ Temp→ pH→ death of pathogen  Transport & delivery to Lab: Prompt.  Processing: immediate/ ASAP (