Lecture Notes on Bacterial Specimen Transport PDF

General Considerations 1. Collect before any antibiotics 2. Collect specimen at actual site of infection 3. Observe aseptic technique in collection of all specimens 4. Consider stage of disease and collect specimen during early stage of illness (2-3 days for viral infections) Typ...

General Considerations 1. Collect before any antibiotics 2. Collect specimen at actual site of infection 3. Observe aseptic technique in collection of all specimens 4. Consider stage of disease and collect specimen during early stage of illness (2-3 days for viral infections) Typhoid fever: Blood culture - (+) first week - Blood culture will be positive for typhoid fever during first week Stool culture - (-) first week - Stool culture will be negative for presence of bacteria during first week 5. Proper instructions to patient must be clear 6. Use proper containers and/or transport media and sufficient quantity of specimen must be obtained 7. Proper labeling with the correct information Level Description Examples 1 critical/invasive Blood, amniotic fluid, CSF, brain, heart valves, pericardial fluid 2 unpreserved/without Bone, feces, sputum, tissue, body fluids (pericardial); preservatives may have normal flora 3 Quantitation required Catheter tip, urine, tissue for quantitation 4 preserved/with Urine in preservative, feces in preservative, swabs in preservatives holding media 5 Batch processing Sputum (AFB culture) Specimen transport Primary objective: - Maintain specimen as near to its original state; maintains viability - Fastidious organism may not survive the nutritionally poor medium - For anaerobic bacteria, transport should not take more than 10 mins - CSF samples - transported within 15 minutes Temperature sensitive: Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae pH sensitive: Shigella Transport Media Stuart’s (1954) 1. Originally formulated for transport N gonorrhoeae 2. Uses charcoal-impregnated swabs which caused difficulty in Gram stain 3. “Non-nutritive” medium. Also as viral transport medium  - Does not have any nutrients to support bacterial growth 4. Good for most specimens, can also be used for stool 5. Some gram-negative rods can utilize glycerophosphate in medium, thus overgrowing culture Amies (1965) 1. Modified Stuart’s medium 2. Replaced glycerophosphate with balanced salt solution 3. Retained charcoal but incorporated it into the medium rather than swab - Charcoal is in the medium, rather than in the swab 4. Better transport system for most specimens (general transport medium) 5. Can be used for respiratory and stool specimens Cary & Blair (1964) 1. Similar to Stuart’s but modified for fecal specimens 2. pH increased from 7.4 to 8.4 3. Removed charcoal from formula 4. Good for stool specimens 5. Recommended for fecal specimens suspicious for Campylobacter spp. And for other enteric pathogens Buffered glycerol saline 1. Designed for stool specimens only 2. Good for mailing fecal specimens 3. High pH to favor fecal pathogens 4. Not for transport of fecal specimens in which Campylobacter spp. Is suspected 5. Toxic to enteric bacteria like Vibrio and Campylobacter Note: Stuart, Amie’s, Cary-Blair Transgrow - for Neisseria John E Martin Biological Environmental Chamber (JEMBEC) - for Neisseria gonorrhoeae Todd-Hewitt and LIM Hanks Balanced salt solution Transport of Specimens: Immediately/Room temperature - Body fluids, bone, CSF, inner ear, corneal scrapings, foreign bodies, gastric aspirate, suprapubic aspirate of urine Within 24 hours/Room temperature - Abscess, outer ear, conjunctiva sample, genital tract specimens (prostate immediately at room temp if in tube), urethra (male) within 2 hours at room temp for JEMBEC system, hair, nails or skin scrapings, respiratory tract specimens, tissue Within 2 hours/Room temperature - Blood or bone marrow Within 24 hours/4C - Rectal swab, stool culture, clean voided midstream urine  Within 2 hours/4C - Indwelling catheter (urine specimen), straight catheter (urine) Immediately/4C - Gastric biopsy Specimen Refrigeration Specimens that can be refrigerated before inoculation of media: - Inoculation of media means introducing bacteria into a culture medium. (ex. Introducing N. gonorrhoeae on CAP) a. Urine b. Respiratory exudates c. stools/feces d. Wounds Specimens that cannot be refrigerated before inoculation of media - Spinal fluids and other body fluids - Genital/cervical for Gonococcus Isolation (swab of - Blood Stored in Refrigerator (4C) Stored at Room Temp Unpreserved urine and feces Abscesses from wound or lesion Sputum Preserved urine and feces IV catheter tips Nasopharynx, Throat Outer ear Tissues, genitals CSF (for viruses) Body fluids Feces for Clostridoides difficile (3 days); more Inner ear than 3 days store at -70C CSF (for bacteria) Specimen Preservation Preservatives Boric acid - Maintain colony count (urine) for 24 hours Polyvinyl alcohol (PVA) and Buffered formalin - Maintain trophozoites and cysts Sodium borate - Recommended preservative for shipping Anticoagulants - To prevent clotting of blood, bone marrow, and synovial fluid (joint fluid)  0.025% Sodium Polyanethole Sulfonate (SPS) - N. gonorrhoeae and some anaerobic bacteria are sensitive to higher concentrations - Peptostreptococcus anaerobius, Gardnerella vaginalis, Streptobacillus moniliformis are sensitive to SPS (even if standard concentrations) Heparin - For viral cultures and tuberculosis - Sodium Thioglycollate Reducing agent to improve recovery of anaerobic bacteria - Agar Small amount provides semisolid consistency to prevent oxygenation and spilling Heparin, EDTA, citrate are the commonly used anticoagulants in blood, however, should be avoided when doing Polymerase Chain Reactions (PCR). For Anaerobic Culture Suitable Unsuitable Blood Sputum, bronchoscopy washings Bone marrow Voided or bladder catheterization urine samples Properly collected abscess material Throat or NPS Lung and transtracheal aspirate Vaginal or cervical swabs Suprapubic urine aspirate Material from superficial abscess or lesions improperly collected Endometrial or endocervical Specimens contaminated with feces, bowel contents, rectal abscesses, draining fistulae Aseptically collected tissue “Sulfur granules” from sputum or when Actinomycosis is suspected Body fluids, CSF, pericardial, pleural, synovial Bile Clinical Specimens Blood Cleansed w/ 70-95% alcohol followed by iodine scrub then alcohol rinse (70-95%Alcohol-Iodine-Alcohol) Avoid normal skin flora while collecting blood from culture Draw 2-3 sets in 24 hours, at least 1 hour apart from the left and right arms  - Ex. draw blood from left arm, wait an hour, draw blood from right arm, wait an hour, draw blood from left arm again. a. Adults: 20 ml per set b. Pediatric: 10-20 ml per set c. Infants and small children: 1-5 ml per set 0.025% SPS (Sodium Polyanethol Sulfonate) Adv. Anticomplementary - Complement system is a system that breakdowns bacteria in bloodstream, inhibiting (anticomplement) the complement system helps preserve bacteria Antiphagocytic - Phagocytosis is destroying pathogens Neutralizes aminoglycosides - Aminoglycosides are a class of antibiotics that can inhibit the growth of bacteria, by neutralizing them, disables their inhibition Disadv. Highest concentration may be toxic Inhibits Neisseria gonorrhoeae, Gardnerella vaginalis, Streptobacillus moniliformis, Peptostreptococcus anaerobius (NGPS) Routine blood culture bottles are held for 7 days (they’re the green/orange/yellow bottles) Signs of growth: bubbles, hemolysis (breakdown of RBC), cloudiness (turbidity), pellicle (accumulation of turbid materials on the surface) Some exceptions are: Brucellosis: 21 days (3-4 wks) Leptospirosis: 6-8 wks Result: conventional - 7-10 days If blood culture is positive, perform Gram stain and subculture media: BAP, CAP, MAC Bacteremia - bacteria in blood, doesn’t multiply Septicemia - bacteria in blood, active multiplication Transient - bacteremia lasts for a short amount of time Intermittent - recurring bacteremia CSF Usually collected using 3-4 tubes 1. Chemistry/Immunology 2. Microbiology, Gram stain, and Culture 3. Hematology 4. Specimen control Rapid testing is recommended (ex. Cryptococcal antigen test, direct stain) Collect at 1 ml and not more than 10 ml total by needle aspiration  Store at room temp. Up to 24 hrs except for viruses which can be held at 4C, for up to 3 days - Note: CSF w/ bacteria: room temp, 24 hrs | CSF w/ virus: 4C, 3 days Cytocentrifuge to collect any sediment for staining and culture Use of acridine orange dye if there is no cytocentrifuge DO NOT REFRIGERATE Common pathogens: Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Streptococcus agalactiae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes (HSNSESL) Acceptable CSF Parameters: No more than 5 lymphocytes/ml Glucose concentration between 45-100 mg/dl Protein concentration between 14-15 mg/dl Testing Media for Specific Pathogens: Cryptococcus neoformans - India Ink method/Latex agglutination Wound and Abscesses (collection of pus) Exogenous wounds - associated w/ traumatic wounds (e.g. gunshot/stabbing), ulcers, burns, animal/human bites, or any foreign body present in skin or mucous membrane Wipe area with sterile solution or 70% alcohol, swab along edge of wound Perform Gram stain Endogenous wounds - associated w/ appendicitis, cholecystitis, cellulitis, dental infections, osteomyelitis, empyema, septic arthritis, sinusitis, and many other INTERNAL infections; many endogenous are nosocomial (LAI) Wipe area w/ sterile solution or 70% alcohol Aspirate material from wall of tissue w/ needle and place in anaerobic transport system Wash any granules and emulsify in saline Both exogenous and endogenous wounds and abscesses infections are commonly caused by anaerobic bacteria so care must be observed in collecting the specimens Media: Anaerobic Blood Agar, MAC, BAP, CAP Throat and Nasopharyngeal Swab Throat swab for Streptococcus pyogenes determination Culture must include anaerobic conditions for beta hemolytic streptococcus Culture on Todd Hewitt Broth for Fluorescence microscopy of beta Streptococcus Use special media for Corynebacterium (cigar) diphtheria, Pertussis, Chlamydia, and Mycoplasma (Neisseria gonorrhoeae if throat) Nasopharyngeal swab - Specimen of choice to detect carrier state of Neisseria meningitidis (enriched CAP)  - Detects presence of Bordetella pertussis - Haemophilus influenzae - Staphylococcus aureus (MRSA) - Corynebacterium diphtheria Best type of swab for throat culture Best swab for genital specimens Most abundant throat flora: Viridans/Alpha-hemolytic Streptococci Most common throat pathogen: Streptococcus pyogenes/Group A beta-hemolytic Streptococci Upper tract - throat, oral cavity, and nasopharynx Lower tract - larynx, trachea, bronchi, and alveolar sacs (to diagnose bronchitis or pneumonia) Media: MAC, BAP, CAP, Amies Throat swab add Modified Thayer-Martin Agar (MTM) and Thioglycolate Broth (Thio) Urine Should be collected in a sterile, screw-cap Specimen of choice: clean catch midstream - Catheterized urine - for those unable to void, allow first 15 ml to pass - Suprapubic urine - 1st morning urine - most concentrated Suprapubic urine - for anaerobic culture Female - Clean area w/ soap and water - Rinse w/ water - Hold labia apart and begin voiding, after several ml have passed - Collect midstream Male - Clean glans w/ soap and water - Rinse w/ water - Retract foreskin, after several ml have passed - Collect midstream Must be preserved or refrigerated Suitable preservative is boric acid to maintain accurate colony count Major cause of UTI: Escherichia coli Colony count should be performed on all urine samples - When performing colony count, use calibrated loop (1 ul or 10 ul) Formula to compute for colony count per ml of urine # of colonies counted x 1000 = colony count/ml of urine # of colonies counted x 100 = colony count/ml of urine Considered significant: >100,000 for UTI / >50,000 CFU/ml - Colony count greater than 100,000 is significant for UTI  - In some cases, it may also be significant when greater than 50,000 < 10,000 CFU/ml - Considered insignificant and may suggest contamination, rather than infection Bacteriuria - bacteria in urine Result - takes about 48 hours to check presence of bacteria Media used: BAP, MAC Sputum Should be collected in a sterile, screw-cap Rinse mouth or gargle w/ water Collected ideally in the morning when it is most concentrated Cough deeply into container May often be contaminated w/ normal flora so it is important to evaluate the quality of specimen Note: number of squamous epithelial cells /LP: 25 Polymorphonuclear Neutrophils (PMN) - acceptable - Fewer than 10 squamous cells, and greater than 25 PMN >25 squamous cell,