Specimen Management & Processing in Microbiology PDF

Specimen Management & Processing in Microbiology Table of Contents LEARNING GOALS & STUDY GUIDE.............................................................................................. ii Specimen Management....................................................................................

Specimen Management & Processing in Microbiology Table of Contents LEARNING GOALS & STUDY GUIDE.............................................................................................. ii Specimen Management........................................................................................................................... 3 Basic Principles of Specimen Collection................................................................................................ 4 Specimen Preservation, Storage and Transport................................................................................... 5 Problems and Solutions................................................................................................................... 5 Safety...................................................................................................................................................... 7 Transport (Shipping) of Hazardous Materials......................................................................................... 7 Specific Specimen Types........................................................................................................................ 7 Aspirates and Tissues...................................................................................................................... 8 Wound Swabs................................................................................................................................. 8 Critical / Invasive specimens.......................................................................................................... 9 Other............................................................................................................................................... 9 Patient-Collected Specimens: Special Considerations...................................................................... 10 Microorganism Viability and Overgrowth............................................................................................ 10 Set-up Prioritization.............................................................................................................................. 11 Requisition and Specimen Information................................................................................................. 12 Unacceptable, Inadequate, Inappropriate Specimens............................................................................ 12 Physical Characteristics of Specimens.................................................................................................. 14 Direct Microscopic Examination.......................................................................................................... 14 Preparing samples for direct microscopic examination.................................................................... 15 Swabs............................................................................................................................................ 15 Fluids............................................................................................................................................ 16 Tissues.......................................................................................................................................... 16 Granules........................................................................................................................................ 16 Examining the direct smear.............................................................................................................. 17 Culture Set-Up...................................................................................................................................... 17 Primary Culture Media..................................................................................................................... 17 Incubation Conditions....................................................................................................................... 18 Sample Processing Techniques......................................................................................................... 18 Non-Routine Specimen Processing............................................................................................... 18 Anaerobic Culture............................................................................................................................. 19 More on Multiplex FYI (Mandell’s 9th)............................................................................................... 20 A Dictionary of Clinical Specimens..................................................................................................... 21 Summary............................................................................................................................................... 24  LEARNING GOALS & STUDY GUIDE On successful completion of this unit the student will be able to: define various clinical specimens received in Clinical Microbiology Laboratory and discuss the basic principles of specimen quality analyze specimen preservation and transport problems for optimal solutions state the two goals of safe practices pertaining to specimens for Microbiology outline transport of hazardous goods regulations for Clinical Microbiology compare different types of specimen collection to the ideal, with emphasis on needle aspirates, tissue samples, wound swabs/ flocked swabs discuss the importance of patient education regarding patient-collected specimens with specific reference to urine, sputum and stool samples recognize common examples of challenges related to organism viability and overgrowth in culture. make correlation between site of collection and most likely bacteria to cause disease when found. apply the four levels of prioritization to specific examples of clinical specimens and state the prioritization of STAT specimens by ward appraise the accuracy, quality and comprehensiveness of specimen information (sample, labeling, test(s) ordered) for appropriate, accurate processing o contrast procedures for unlabeled/ mislabeled for invasive vs. non-invasive specimens o describe inadequate/inappropriate specimens using common examples correlate physical characteristics derived from gross examination of specimens with the implications for the lab when processing those specimens discuss the role of direct microscopic examination: describe ways in which a direct smear is useful; detail situations in which a direct smear is of limited usefulness and recognize situations in which a technique other than Gram-stain is advantageous relate the method chosen for making an optimum direct smear to the following specimen types: swabs, liquids (consider viscosity and volume); tissues; granules outline the purpose of using a battery of media in set-up of specimens with reference to the different classifications of media and their purpose(s) discuss the challenges inherent in anaerobic culture: atmospheric requirements from collection to inoculation; obtaining an optimal specimen recognize contraindications for anaerobic culture requests ii Specimen Management The following “Ten Points of Importance” are adapted from Miller et al., CID 2018:67 1. Specimens of poor quality must be rejected. 2. Labs should not be demanded to report “everything that grows.” 3. Sites with commensal microbiota require care in the collection of specimens. 4. A specimen, not a swab of a specimen, is the specimen of choice. 5. “The laboratory must follow its procedure manual or it will face legal challenges.” 6. Collect specimen(s) prior to administration of antibiotics whenever possible. 7. Susceptibility testing is only performed on clinically significant microorganisms. 8. Lab-reported results should be accurate, significant, and clinically relevant. 9. The lab should set technical policy; not the purview of the medical staff. 10. Label specimens accurately and completely. It cannot be over-stated that results will only be as good as the specimen. A quality specimen encompasses: proper specimen collection, including patient-collected specimens complete and correct labelling issues specimen suitability for the analysis required, including sufficient volume contamination issues specimen preservation, storage and transport Although it is not possible to verify all the pre-examination steps leading to arrival of a specimen in the lab, laboratory personnel must be aware of the possible complications and effects on results. Pre-examination steps (pre-analytical) upon arrival in the lab: specimens are prioritized – deal with STATS and fragile specimens asap specimens are accurately identified and matched to requisitions (if a requisition has been sent) or, if there is no paper work, the order in the laboratory information system (LIS) must be checked quality and suitability of the specimen relative to the test requested is considered supplementary necessary steps (usually phone calls to the ward or doctor’s office) are determined and acted on to ensure that additional tests are ordered, or more appropriate specimens are collected/recollected as deemed necessary cost-effective approaches are inherent in the pre-examination phase, limiting the frequency of repeat specimens and culture as well as selective culturing in some instances, a portion of the specimen must be forwarded to another lab. Examples: o sputum for tuberculosis (TB) to BCCDC o bronchoalveolar lavage (BAL) /brushing for cytology to Histology o tissue for cytology to Histology o CSF [four tubes are collected]: Tube 1 to Chemistry; Tube 2 to Microbiology; Tube 3 to Hematology, Tube 4 for other special studies or is a control tube. On entering the examination phase (or analytic phase), suitable staining technique(s), media and incubation temperature/atmosphere are determined by both the specimen source and diagnosis. Following specimen processing, the remaining portion of the specimen is stored for a specific time at a specific temperature. Retrieval of stored specimens is important for the post-examination phase (post-analytic) where the final culture report is sent to the doctor, interpreted and acted upon. Proper storage of the original specimen makes it possible to repeat a culture or smear, add tests, investigate for infection control/epidemiology purposes, or to clarify clerical issues. See https://www.mountsinai.on.ca/care/microbiology/clinical-laboratory/specimen-collection/bacteriology for an example of a laboratory specimen collection guideline. 3  Basic Principles of Specimen Collection Take specimens in the acute phase AND prior to antibiotics. knowledge of pathophysiology for optimal collection time e.g. blood in the first week of illness and feces or urine in weeks 2-3 in typhoid fever highly susceptible organism(s) may not be recovered post-antibiotics if post-antibiotic: accept specimen (does not necessarily preclude organism(s) recovery, but add a comment that viability/recovery may be compromised) Label specimen accurately and completely. patient’s full name, MRN, date & time, source, ordering clinician most importantly, signature/ initials of collector on the specimen label affixed directly to the specimen Collect sufficient quantity for analysis using appropriate collection technique. appropriate site (actual site of infection) avoid contamination by normal flora (particularly a problem with swabs) appropriate volume – 1 drop per plate/broth culture therefore 0.5 ml considered a standard minimum for liquids and 1 cm3 for tissue Use appropriate collection devices, containers. sterile containers, single swab vs. dual swab format in transport media, flocked swab advantages, anaerobic transport systems, fluid and tissue will be detailed Written requisitions should indicate all pertinent information including a full and accurate description of the specimen site as well as time of collection in addition to patient information and ordering doctor. “Dog bite wound Rt forefinger” is meaningful. “Wound” is not. if multiple samples are received from the same patient, each of the specimens must be identified accurately e.g.: o leg ulcer (L thigh); leg ulcer (R calf) o pre-prostatic massage, post-prostatic massage, clean catch urine information included with the specimen guides culture and interpretation - see “Requisition and Specimen Information” on Pg 12 Transport safely and promptly. most specimen types should be transported at room temperature with an optimal transport time of < 2 hours without some sort of preservation (some specimens will not tolerate any delay) and in general, a maximum of 24 hours with preservation (e.g. refrigeration or holding medium) avoid hazards from leakage for molecular assays, check manufacturer recommendations – freezing at -80° may be optimum if delays fresh culture material is essential: in the case of fluid samples, slow-flowing lines do not provide suitable specimens due to possible overgrowth of slow-growing, fastidious organisms by any rapidly growing organisms that might also be present o urine from urine collection bags is NOT a fresh sample – unacceptable o swabs are collected into transport media to slow down rapid multiplication where delays in transport may be unavoidable The lab should be notified if unusual pathogens or risks are suspected e.g. TB positive patient, rabies exposure, Brucella, etc. Fall 2024 4  Specimen Preservation, Storage & Transport The goal is to maintain the specimen as near to its original state as possible - minimal deterioration of any pathogen(s) with minimal overgrowth by contaminants - while preventing risk to personnel. Problems & Solutions The following can all affect the recovery of microorganisms and the validity of the culture report: − overgrowth − accurate quantitation of organisms − drying − morphology − temperature − oxygen exposure − pH − clotting Solutions to address the challenges of transport delay use temperature, preservatives and/or media, as appropriate, to the needs of a variety of cultures. Pre-examination Storage When delay is unavoidable prior to set-up: a 2-hour maximum delay is recommended for most fluids some specimens may be refrigerated for up to 24 hrs: o IV catheter tips, viral study CSF, outer ear swab, unpreserved feces, sputum, unpreserved urine many specimens should be held at room temperature, NOT refrigerated, to preserve pathogens that do poorly at lower temperatures: o any specimen for anaerobic culture, body fluids, inner ear, eye, preserved feces, genital, nasal/ nasopharynx, throat, tissue, preserved urine blood cultures should be placed in an incubator @ 35°C asap small volumes ( Salmonella coliforms (Enterobacterales) Anaerobes Staphylococcus Mycoplasma Legionella Chlamydia Mycobacteria Viruses Yeast unpreserved parasites Fungi toxin from Clostridiodes difficile From Mahon, C. & Manuselis, G. Diagnostic Microbiology, 2nd. Set-up Prioritization The following four-level prioritization does not cover all possible situations. Priority levels reflect severity of disease/ invasiveness of collection procedure. Urines may be refrigerated up to 24 hours prior to processing. Blood cultures: hold at RT until loaded onto instrument. If held in incubator, a manual workflow should be followed. In addition to the specimen type, consider the urgency of the request. STATS should be prioritized by ward: OR > ICU > ER. (MCM 11th ed. recommendation) The turn-around-time for a STAT on a direct smear is about 30 minutes, even less for CSF specimens. Levels of Specimen Prioritization Level Description Specimens 1 Critical/Invasive Amniotic Fluid Blood Brain & CSF Heart valves, Pericardial Fluid 2 Unpreserved Body fluids other than those above Bone Drainage from wounds Stool Sputum Tissue 3 Quantitation Catheter tip Urine Tissues for quantitation 4 Preserved Stool in preservative Urines with preservative Swabs in holding media (aerobic & anaerobic) Mahon & Lehman, Diagnostic Microbiology, 6th ED 2019 Fall 2024 11  Requisition and Specimen Information The requisition (electronic or paper) should supply the specimen source, diagnosis, ± history and test ordered in addition to all of the labelling information (see below). Patient: name, age (DOB), and gender; room number or location Physician: name and address Source (specific anatomic site): o specific e.g. not simply “skin” or “wound”, but should include description of site e.g. “Posterior right knee incision site” (“Post. Rt. knee-incision”) Date and time of collection Diagnosis/history: o guides choice of media, incubation temperature, atmosphere and duration o directs level of identification (genus & species, or only to genus level) o determines antimicrobials to be tested Antimicrobials if patient is currently being treated Name of healthcare worker transcribing orders Test: C&S is the routine request for “bacterial culture and AST” and should be specified. Examples of multiple testing that could be ordered on a single sample: o The diagnosis is “gastroenteritis” and a stool specimen is sent to the lab. Is it for C&S? O&P? viral studies? all of the above? o BAL often collected for all of the following: C&S, AFB, fungi, cytology Labels should provide all the specific information as above either in the scan-able barcodes or in paper format. patient name, patient identification (chart number, birth date), room number, ordering clinician, source of specimen, time of collection. should also show who collected the specimen - on the sample even if nowhere else Most hospital labs now use bar-coded wristbands and scanner/ label printers at the point of collection which solves most issues (orders can be accessed at bedside ensuring collection matches order) but could introduce other issues (positive patient identification must still include a verbal). Unacceptable, Inadequate, Inappropriate Specimens Sometimes specimens “cannot be accepted due to inability to correctly interpret the results” (MCM 11th p. 285), i.e. specimens have to be rejected, and should be recollected ASAP. Keep in mind that some specimens are extremely invasive/ critical and cannot be replaced e.g. CSF, bone marrow, surgical/ tissue biopsies. These are NEVER rejected. In all instances, a phone call to the collecting/ in charge individual is required. Never discard before having spoken to a healthcare team member. If the specimen is rejected, document the reason. If the physician insists on processing, add a cooment to the report regarding possibly compromised results. (Mahon 5th) Fall 2024 12  If the information on the specimen does not match the requisition (mislabeled) or the specimen is unlabeled: Phone the ordering physician/ward as soon as possible and if a(n): o invasive specimen: do not proceed with processing the specimen until the “collecting” person makes necessary corrections to the specimen - in person, in the lab; if this is not possible, should be dealt with through the supervisor o non-invasive specimen: ask for a “recollect”, explaining why this is necessary – reject, do NOT process Examples of problems that may give rise to specimen rejection PROBLEM EXPLANATION leaking specimen may be contaminated and/or potential health risk to staff syringes with needles syringes should have the needle removed and be capped; attached usually critical/ invasive specimens - not rejected but may be unsuitable for anaerobic culture stools contaminated with interfere with culture urine, barium, oils unpreserved specimens held overgrowth of hardy organisms and poor survival of fragile at room temperature for >2 organisms hours inappropriate specimens for see Level 2 anaerobe notes anaerobes dried-up specimens such as vegetative cells need moisture those collected on out-of-date swabs specimens in formalin, purpose is to be antiseptic/antibacterial! mouthwash a single swab or small e.g. a Dr. requesting aerobic & anaerobic culture, fungus volume sample is sent for and “AFB” on < 0.5 ml of aspirate should be called to multiple tests determine which testing is the priority, the other orders rejected and additional specimen requested a charcoal swab is require a plain (direct smear) plus a charcoal swab inappropriate for direct (neutralize toxic substances) smears anaerobic cultures from sites commensals confound results - reject containing mucosal normal flora or contaminated with feces frequently ordering tests cost-effective use of lab services limits frequency of tests within a stated time period and can result in rejection of what are essentially duplicate specimens Fall 2024 13  Physical Characteristics of Specimens Gross examination of the specimen also provides useful information about the physical characteristics of the specimen. Document the physical characteristic. Characteristic Why might this be important? swab or aspirate? stool: formed or liquid, bloody? swab or fluid is bloody? small volume received? fluid is clear or cloudy? pus, gas, foul smell? sulfur granules? Direct Microscopic Examination The majority of routine culture types incorporate a Gram-stained “direct smear.” Direct smears are made directly from the patient specimen, not from culture growth, and can provide valuable information to guide treatment prior to culture results. In specific instances, unstained preparations (e.g. negative stain, darkfield exam, wet mount for motility, iodine mounts for some parasitology, KOH mounts for fungal elements in keratinous specimens) are more useful than stained preparations. However, direct smears are not useful from certain specimens: Direct smears are NOT typically included if: the pathogen microscopically resembles the normal flora o Examples: throat, cervix (however, a cervix ds/ may be read for presence of polys i.e. PMNs), stool or rectal swab turn-around time/pathogenicity is low o Example: nose very small numbers of organism, making direct smears an inefficient procedure o Examples: urine, IUD, catheter tip (also, direct smear of IUD or catheter tip requires using a small amount of saline which is then applied to the slide which further dilutes) FYI: If specifically requested by the physician on the specimens noted above, labs will usually perform a direct smear. Fall 2024 14  Prompt reading of the direct smear, regardless of the staining technique (stains other than Gram-stain are occasionally used) serves various purposes: determines the quality of the specimen o Sputum “Q score”: The ratio of squamous epithelial cells (SECs) to polymorphonuclear cells (PMNs/ WBCs) is compared. Sputum is from deep in the lungs. If there is a predominance of squamous epithelial cells (SECs) when performing the Q score, the specimen is considered salivary (URT), not sputum (LRT) and the culture should not be interpreted. If the patient is immunocompetent and PMNs predominate, the sample has been coughed up from deep in the lungs and the culture is interpreted. (This scoring system does not apply to immunocompromised patients.) acts as a diagnostic aid o BV scoring for diagnosis of bacterial vaginosis is done from a smear o Neisseria gonorrhoeae STI can be diagnosed on a male urethral direct smear o viral vs. bacterial infection (if bacterial, PMNs are normally present and, based on the Gram-stain result, the physician may be able to initiate antibiotics without waiting for culture results; antibiotics are ineffective against viruses) additional, non-routine processing may be suggested o adding anaerobic, TB or Mycology culture to routine C&S based on unanticipated microscopic elements seen in the direct smear guides culture interpretation by the technologist o relative number of organisms seen o presence or absence of PMNs o microorganisms seen in smear fail to grow in culture: Why? Were they non- viable or, was routine culture inadequate for their recovery? Preparing samples for direct microscopic examination Samples may be examined by “wet prep” (fluids or semi-solids), cytocentrifugation (clear to slightly turbid fluids), or smear. The method of preparing the smear relates primarily to the sample e.g. thin or thick fluid, granular or mucoid fluid. Regardless of the method chosen, the goal is to produce both thick (not opaque!) and thin (monolayer) areas. The best portions of a sample to investigate for infection (for smear and culture set-up) will have evidence of all or some of the following: pus (yellow to tan), blood, necrosis (blackening), mucus. Swabs best practice: one swab for culture, the order for the smear (the more plates being used, the more important this is) rolled rather than smeared, with all sides of the swab in contact with the slide o rolling better preserves morphology and better represents the relationships of microorganisms and cellular elements in the sample Fall 2024 15  Fluids Single layer: If thick, viscous or cloudy, apply a single drop (viscous fluids may lift and wash off, methanol fixing decreases this likelihood compared to heat-fixing). Layered smear: If clear and small volume or small numbers of organisms anticipated, apply a drop, allow it to dry, apply a second drop. Sediment: If a large volume of fluid, centrifuge at 1500g for 15 minutes, remove supernatant (may need to save supernatant) leaving sufficient volume (usually 0.5 ml) to: re- suspend sediment; inoculate all plates; prepare extra smears. This method is typically used with dialysates, pleural and peritoneal fluid and CSF in labs lacking a cytospin or other concentration instrumentation. Cytocentrifugation (Cytospin): This instrumentation concentrates a small amount of fluid directly on a slide while blotting excess liquid – ideal as it concentrates microscopic material in a small area and maintains morphology. This method is typically used with CSF. Micropore Filtration: Used if a large volume of fluid to produce a sediment. FLUID: Fluid:cytocentrifuge DROP (Single layer OR 2-Drop) Tissues Touch-prepped: Pick up tissue with sterile forceps and touch to media and slide(s). Ground or minced: Sterile single-use grinders are commercially available to grind tissue. Mincing is accomplished using forceps/scalpels or scissors which are in sterile packs at the set-up bench. Emulsify the tissue in a small amount of sterile saline and use this for both the smear and media inoculation. Granules Place the granules between two labeled slides and crush by pressure with rotation to distribute the material. Stain the best slide. Fall 2024 16  Examining the direct smear Evaluate on low power to determine homogeneity of the preparation. This assists in picking the most representative area to read. Normally, ten good fields should be assessed and averaged when on 100x oil. Assess for presence of contaminants (normal flora, squamous epithelial cells, food or other debris, bacteria but no PMNs). Be aware of other “clues”: Ciliated columnar epithelial cells, alveolar macrophages (dust cells), and Curschmann’s spirals in a sputum confirm that the specimen is from the lower respiratory tract i.e. a true sputum and not saliva. Elastin fibers are often present in necrotic tissue. Culture Set-Up Primary Culture Media Media selection is appropriate to the specific requirements of a specimen type and diagnosis. Example Classification/purpose non-selective every routine set-up will BAP: Columbia agar with Sheep’s Blood BAP or TSA with include sheep’s blood or Sheep blood base agar enriched if fastidious organisms are Chocolate (CHOC) potential pathogens if a sterile site Add Brucella (BRUC) anaerobically to BAP and CHOC if small #s of potential - Brain Heart Infusion Broth with Fildes (BHIB/w Fildes) for CSF pathogens & sterile site - THIO or Cooked Meat Broth for anaerobes selective if pathogens may be - Colistin Nalidixic Acid (CNA) to inhibit Gram-negative coliforms overgrown by organisms and select for Gram-positives normal to that site - PEA similar but used more for recovery of anaerobic Gram- positive organisms - Brucella with Neomycin (BRUC/Neo) to inhibit Gram-negative facultative anaerobes - MacConkey (MAC) to select (and differentiate) Gram-negatives selective for organisms OTHER than bacteria (yeast, molds) if slower growing than Potato Dextrose agar (PDA), Sabouraud Dextrose Agar (SAB = normal flora in a site SDA), Nickerson-Bismuth Sulfite-Glucose-Glycine-Yeast Agar (BiGGY) for yeast selective and differential if small numbers of - stool culture: some or all of: MAC, MacSORB) Hektoen Agar, pathogens must be Deoxycholate Citrate Agar), Cefsulodin-Irgasan-Novobiocin, screened from large Campylobacter Media etc. etc. numbers of normal flora - cervix culture Thayer-Martin (TM) or Martin-Lewis (ML) enrichment if small numbers of - Selenite Broth and Gram Negative Broth for the recovery of pathogens must be enteric pathogens particularly Salmonella and Shigella increased relative to large numbers of normal flora Fall 2024 17  Incubation Conditions Selection of incubation conditions (temperature, atmosphere, duration) are based on the growth requirements of pathogens suspected in a body site. increased CO2 is used for fastidious and capnophilic organisms, typically for upper and lower respiratory (URT, LRT), genital, body fluids and wounds routine incubation temperature is 35-37°C o Campylobacter at 42°C o Molds at room temperature, yeasts at 35-37°C Routine aerobic plates are examined (“first read”) after 16-18 hours’ duration. Most are re- incubated and re-examined at 48 hours, sometimes at 72 hours as well. anaerobic plates are incubated an initial 48 hours before reading enriched broths are examined daily and held for 5-7 days enrichment broths have shorter incubation times, as little as 4 hours, which should be adhered to as inhibitory agents become increasingly inhibitory for the pathogens as well extended incubation or addition of more enriched media: organisms seen in direct smear not growing using routine conditions and media; physician’s request to cover for unusual pathogens Sample Processing Techniques Ideally, specimens should be in liquid form to allow equal distribution to each media. liquefaction involves placing a swab into 0.5 - 1.0 mL broth, vortexing and dispensing with a sterile pipette; this technique was not common for practical reasons (time!) with the move to automated inoculation and streaking platforms (WASP, Kiestra InoqulA) and flocked swabs (BD ESwab™, Copan™), samples can be eluted to provide specimens in liquid form (elution) tissue may be ground in sterile grinding kits with a small amount of broth or saline, or “stomached” in a Stomacher Lab Blender filtration may be used to process large volumes e.g. Continuous Ambulatory Peritoneal Dialysis (CAPD) dialysate (see previous notes on direct smears). o alternatively, centrifugation may be used to concentrate (sediment a fluid) when small numbers of organisms are significant. Non-Routine Specimen Processing Non-routine specimens offer challenges to proposing and implementing standardized procedures − multiple lumen catheters − intra-uterine devices (IUD) − vascular catheter tips − implant soak solutions − veins and heart valves (natural and − sterility specimens (pharmacy and prosthetic) environmental) Where a standardized procedure does not exist, questions such as those below (based on Mahon & Manuselis 5th ed. p. 123) help to guide processing. Low or high numbers of organisms expected? (Is concentration of a large volume an asset?) Is a low number expected and is enrichment an asset e.g. sterility checks where even one colony is significant? Fall 2024 18  Are fastidious or non-fastidious isolates likely? Is normal flora also present? Inhibitory media and timely processing is important. Might preservatives or inhibitory substances be associated with the sample? Dilution might help. What is the best representation of a sample - how much must be cultured? e.g. a 12 cm piece of vein might have only a single small infected site (a “plaque”). Is a specific infectious agent in question? Does the sample have inner and outer surface that must be cultured e.g. catheters? Anaerobic Culture if anaerobes are suspected, collect an optimal specimen and transport quickly tissue and fluids/aspirates are optimal specimens for anaerobic culture sterile saline should be added to small tissue samples to prevent drying and processed immediately uncapped needle aspirates for anaerobic culture should be injected into an anaerobic transport system for transport to the lab Why the hurry? o anaerobes range from aerotolerant (will grow poorly in a CO2 incubator) to moderate (can survive in the presence of 2-8% oxygen but cannot reproduce if levels exceed 2.8%) to the strict anaerobes (die if exposed to room oxygen for 10 minutes and cannot reproduce if residual oxygen exceeds 0.5%) o anaerobes are very susceptible to drying Would you refrigerate or keep at room temp? o room temp! - refrigeration can oxygenate the specimen Swabs are not ideal - why? o if wrapped fiber swabs, the fibers trap organisms o drying o contact a large surface area, more likely to be contaminated with superficial normal flora when attempting to sample a deep (read this as “anaerobic”) infection Anaerobic transport systems: various are available but all strive to minimize exposure to oxygen: pre-reduced media; gassed out vials; anaerobic pouches Specimen suitability for anaerobic culture: o sterile sites/ body fluids or walled-off abscesses are suitable o specimens likely to be contaminated esp. with normal anaerobic and facultative anaerobic flora are not suitable media may be purchased as PRAS = pre-reduced and anaerobically sterilized Fall 2024 19  plated media is placed in anaerobic incubation conditions ASAP Primary Anaerobic Culture Media: A battery of plates is typically used anaerobically; minimal set-up would be just a BRU/ANO2. Some examples of media: o non-selective: Brucella (BRU) or any media with hemin and Vitamin K1 added o selective: PEA or CNA, Laked Blood w/ Kanamycin and Vancomycin (LKV) o a thioglycollate broth may be added if bacterial load is expected to be light (contains reducing agents thioglycollate and L-cystine plus a small amount of agar to reduce convection currents) OR a cooked meat broth (latter is better for longer storage of anaerobes because the meat acts as both a reducing and detoxifying substance, unlike the thio which lacks neutralizing substances and becomes toxic to anaerobes over time) More on Multiplex FYI (Mandell’s 9th) “A multiplex polymerase chain reaction panel for testing CSF (FilmArray Meningitis/Encephalitis panel; BioFire Diagnostics LLC) has been approved by the FDA. The panel includes 14 targets (Escherichia coli K1, H. influenzae, L. monocytogenes, N. meningitidis, S. agalactiae, S. pneumoniae, cytomegalovirus, enterovirus, HSV types 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, and Cryptococcus. neoformans and C. gattii) and generates results in approximately 1 hour… Detection of viruses in respiratory samples has changed with the introduction of FDA- approved multiplex NAATs that detect 17 or more respiratory viruses. These panels have largely replaced culture-based and antigen-based tests for viral detection in respiratory samples. The preferred sample is a swab passed through each nostril all the way to the back of the nasopharynx and placed into viral transport medium… The specific organisms included vary and include adenoviruses, coronaviruses, human bocavirus, human metapneumovirus, influenza A virus (including subtypes H1, H3, and 2009 H1N1), influenza B virus, parainfluenza virus 1– 4, and respiratory syncytial virus (including A and B); bacteria including Chlamydia pneumoniae, M. pneumoniae, Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, and Bordetella holmesii are included in some panels. NAAT performed on a posterior nasopharyngeal swab or aspirate is the recommended test of choice for B. pertussis and B. parapertussis detection. Although B. pertussis can be cultured on specialized media, culture is less sensitive than NAATs… There are currently multiple FDA-approved multiplex NAATs that simultaneously detect five or more stool pathogens… The Luminex GPP detects Campylobacter species, C. difficile, E. coli O157, enterotoxigenic E. coli, Shiga toxin–producing E. coli, Salmonella species, Shigella species, Vibrio cholerae, adenovirus 40/41, norovirus, rotavirus A, Cryptosporidium species, Entamoeba histolytica, and Giardia species in approximately 5 hours. The BioFire GI Panel can identify 22 targets in approximately 1 hour, and targets include all of those on the Luminex GPP plus Vibrio species, Yersinia enterocolitica, enteropathogenic E. coli, enteroaggregative E. coli, Plesiomonas shigelloides, astrovirus, sapovirus, and Cyclospora cayetanensis; Shigella species and enteroinvasive E. coli are targeted instead of Shigella species.” Fall 2024 20  A Dictionary of Clinical Microbiology Specimens BODY FLUIDS Amniotic fluid — fluid produced by the innermost layer of the placenta early in gestation and contained within the amniotic sac surrounding the embryo in utero. Ascitic fluid — serous fluid aspirated from the abdominal cavity (the peritoneum). Bile — a brown-green fluid secreted by the liver and either poured into the intestine or concentrated in the gallbladder. Bone marrow — the soft, highly cellular, blood-forming tissue that fills bone cavities. Joint (synovial) fluid — alkaline, thick fluid contained in joint cavities, bursae, and tendon sheaths serving as a lubricant. Pericardial fluid — fluid contained within the membranous sac that encases the heart. Peritoneal fluid — same as ascitic fluid. Pleural fluid — fluid within the thoracic cavity which encases the lungs. Spinal fluid — fluid contained within the membranous coverings of the spinal cord and brain within the space known as the subarachnoid space. Transudate — fluid which has passed through a membrane or extruded from a tissue and characterized by its low viscosity, lack of protein, and cells or cellular debris, and by having a specific gravity under 1.013. EYE Conjunctiva — the mucous membrane covering the anterior surface of the eyeball and the under surfaces of the eyelids. Inner canthus - the inner (nasal) angle formed by the union of the upper and lower eyelids. Lid — folds of skin that protect the anterior eyeball surface. GENITAL (FEMALE) Bartholin’s gland — one of two, small, mucous-secreting glands on either side of the vaginal orifice. Cervical aspirate — mechanical withdrawal of material from the cervix. Culdocentesis — aspiration of fluid from recto-uterine excavation by puncture of the vaginal wall. Endocervical — from the interior of the cervix. Endometrium — the mucous membrane comprising the inner lining of the uterine cavity. Fallopian tube — tube from uterus to ovary. 4 7 Female genital — nondescript term generally taken to mean a vaginal/cervical specimen. Lochia — the final vaginal discharge occurring 1-2 weeks after childbirth. Ovary — reproductive egg-forming gland within the pelvis in the female, lying lateral to the uterus. Perineum — the space between the anus and the scrotum of the male and the anus and vulva of the female. Placenta — highly vascularized organ of pregnancy, composed of multiple layers within the gravid uterus, supplying nutrients and gas exchange to the fetus. Placenta (C-section) — (see placenta) a result of a cesarean section. Fall 2024 21  Urethral — from the membranous canal conveying urine from the bladder to the exterior of the body. Uterus — hollow, muscular organ in the female in which the fetus develops. Vaginal — from the canal that extends from the vulva to the cervix. Vulva — the region of the external female genitals. GENITAL (MALE) Lesion — a circumscribed pathologic or traumatic injury to tissue. Penile exudate — exudate expressed through the urethra. Prostate — a gland which, in the male, surrounds the neck of the bladder and the urethra. Urethral — see above. LOWER RESPIRATORY TRACT Bronchial — referring to the large air passages which dichotomously branch within the lungs. Bronchial aspirate — material collected from the bronchi by means of instrumentation. Fiberoptic — collection of material with an instrument designed for visualization of the lower respiratory area and for specimen collection. Sputum - matter ejected from the lungs, bronchi, and trachea through the mouth. Tracheal — the tube from the larynx to the bronchi. Transtracheal aspirate — material obtained by surgical passage of a catheter through the tracheal wall and into the lower respiratory area. UPPER RESPIRATORY TRACT Ear — unless specified, refers to the external ear. Mouth and dental — gums, gingivae, teeth, root canals, tongue, etc. Nasopharynx — that part of the pharynx above the soft palate. 48 Nose (nasal) — in microbiology the term usually refers to culture obtained from about 1-2 cm deep within the nostril. Sinus — any body cavity, hollow space, or open channel. Throat — that area within the deep oral cavity between and including the tonsillar pillars from where cultures are obtained. STOOL/RECTAL — a term referring to the fecal discharge from the bowels. SURFACE SPECIMENS Burn — traumatic lesion caused by contact of tissue with heat. Cyst — any liquid- or exudate-containing sac. Decubitus — ulceration due to prolonged pressure of lying down or sitting. Exudate — fluid containing protein, cells or solid material which has escaped from blood vessels as a result of injury or inflammation. Laceration — a cut. Lesion — see GENITAL (MALE). Paronychia — inflammation involving the folds of skin around the fingernails. Skin — external body covering. Fall 2024 22  Stoma — any small opening or orifice on a free surface, i.e. the opening from a colostomy or ileostomy site. Suture — a surgical “stitch.” Ulcer — a loss of integrity of a cutaneous or mucous surface lining resulting from the sudden or gradual sloughing of necrotic tissue. Vesicle — a small blister containing a serous liquid. SURGICAL SPECIMENS Abscess — localized collection of pus in a cavity formed by disintegration of tissue. Aspirate — removal of fluids from a cavity by suction. Biopsy — surgical removal of small portions of tissue from a living body for the purpose of establishing a precise diagnosis. Bone — mineralized connective tissue that makes up the skeleton of vertebrates. Clot — a semisolid mass, usually of blood or lymph. Drain — an artifically placed device used to create a channel by which fluid or pus can be exited from a cystic space or body cavity. Exudate - see SURFACE SPECIMENS. Fistula — an abnormal passage or communication between two organs or to the outside. Hematoma — a tum or of effused blood - a bruise. IV catheter — tubing used to infuse sterile material into the veins. Prosthesis — an artificial body part. Pus — a liquid inflammatory product of leukocytes and fluid. Stone — a very hard mass or calculus usually composed of mineral salts. Tissue — a surgically removed mass of body cells. Wound — see lesion, GENITAL (MALE). URINE Catheterized — urine aspirated from a urinary catheter. Midstream — urine collected in a container after the first few milliliters of urine has been passed. Suprapubic — urine surgically aspirated with syringe and needle by direct puncture into the bladder. Reference: Specimen Management tin Clinical Microbiology Miller, J. Michael (Jon Michael) ASM Press, 1999 Fall 2024 23  Summary It is useful to consider the diagnostic cycle in Clinical Microbiology in three phases (adapted from Koneman’s 7th ed.): pre-examination o patient goes to the doctor with signs and symptoms of infection o tentative diagnosis o specimen collection o relevant test orders e.g. C&S and prompt transport to lab o data input on receipt in the lab examination o direct examination of specimen ± direct smear ± presumptive report o suitable culture (inoculation, media/ atmosphere/ duration) o culture examination and testing (ID tests and AST) o interpretation ± subculture ± preliminary report post-examination o formal report to physician o choice of therapy by physician The course to date has focused on the examination or “C” (culture) phase of the cycle but has also included pathogenesis and disease pre-examination as well as an introduction to reporting and treatment options post-examination. The unit on anaerobes stressed the importance of suitable specimens, and this module will look more closely at the pre-examination phase. Results are only as good as the specimen. What constitutes good specimen collection? Sampling the right site adequately and correctly and at the right time, LABELING completely and correctly, transporting safely and promptly, and alerting to any special requests or special risks. Particularly with lab centralization, safety and transport concerns are paramount. Viability and overgrowth solutions include refrigeration, preservatives and holding media. The best specimens (tissue and aspirates) are also the most invasive. Understandably, swabs are very common. Patient- collected specimens present unique challenges for adequate sampling. Prioritization on arrival at the lab ensures the most critical specimens are dealt with first. The lab may reject specimens if they would cause an “inability to correctly interpret the results.” This could be a result of labeling errors or inappropriate specimens. However, invasive specimens e.g. CSF and biopsies should never be rejected. Communication with other health care professionals is vital. Gross examination of specimens is important as physical appearance can suggest additional investigation or conversely, suggest that results may be compromised e.g. swab vs. aspirate. Direct smears may be useful in providing meaningful, timely information that can guide treatment. Some direct smears are diagnostic (BV scores on vaginal specimens) while other direct smears can be used to assess specimen quality (Q score on sputum). A variety of techniques are used for preparing and staining direct smears based on the type of specimen (swab, fluid, tissue) and what is suspected (Gram’s stain routinely but also e.g. AFB, fungus stain). Routine culture set-up considers the requirements of expected pathogens and the likelihood of normal flora. Culture may be as simple as a single BAP (as in a throat swab) or as extensive as multiple primary media that includes enriched, selective, differential, enrichment or enriched broths. Incubation may be as little as 16 hrs hours or as long as weeks. The laboratory set-up manual provides details for almost every specimen type received in the lab. Fall 2024 24