The clinical microbiology laboratory offers an increasingly diverse menu of diagnostic assays, including maintenance of traditional methods first developed in the early 1900s, to implementing increasingly complex molecular assays and, most recently, implementing whole-genome and next-generation sequencing assays for pathogen detection, typing, and assessment of antimicrobial susceptibility. The laboratory is also becoming increasingly automated, from the adoption of automated enzyme immunoassay (EIA) processors, to nucleic acid extraction and amplification platforms, to the implementation of total laboratory automation systems. Regardless of the diagnostic method, the accuracy and relevance of results generated by the microbiology laboratory are dependent on multiple factors, including whether the right specimen was collected and transported to the laboratory appropriately, whether the correct test was ordered for that specimen type, and when the specimen was collected relative to patient presentation and antibiotic initiation, among others. This chapter aims to provide general commentary regarding important features of specimen collection, to present the advantages and limitations of common diagnostic assays, and to address a number of common questions and points of confusion among health care workers.
Specimen Collection: What, When, and From Where
Specimen Collection and Preanalytic Variables in Microbiology Testing
Proper specimen collection and transport to the laboratory are critically important for appropriate patient management. Submission of poorly collected specimens may lead to failure to recover or detect significant pathogens. Results may be difficult to interpret when specimens are improperly collected due to recovery of colonizing, nonpathogenic organisms. If a laboratory performs testing on a specimen that experienced prolonged transport time or was submitted under improper transport conditions, false-negative or false-positive results are likely. Downstream consequences may include an inaccurate diagnosis and/or inappropriate treatment. Administration of unnecessary treatment or procedures may in turn result in increased length of hospital stay, drug side effects, and/or additional costs.
Rejection of specimens may be due to improper sample labeling, delay in transit to the laboratory, collection in an improper container, or improper specimen type. As testing menus vary among laboratories, so do the specimen collection devices and guidelines. Differences in laboratory collection guidelines are due in part to the differences in the types of assays performed. Test manufacturer’s guidelines specify the type of specimen validated for that particular assay, and laboratories should adhere to these guidelines. Therefore clinicians should be familiar with the specimen collection guides of their laboratories. Such guides often outline collection instructions, transport conditions, volume of specimen required, and rejection criteria. General specimen collection and transport guides focused on microbiologic tests are also available. Table 2.1 outlines examples of specimen collection and transport guidance based on various body sites.
|Specimen Type||Collection Guidelines||Transport Guidelines||Comments|
|Abscess, including wounds||Cleanse surface with sterile saline or alcohol. Do not submit swabs.||Utilize anaerobic transport system for anaerobic cultures.||Tissues or aspirates are superior to swabs. Contamination with surface material may occur with colonizing organisms unless the site is well cleaned.|
|Blood||Collection of serum, plasma, or whole blood may differ depending on testing requested. Disinfect culture bottle top and patient skin.||For blood culture bottles: as rapidly as possible; within 2 hours is optimal.||Collect more than one set, including aerobic and anaerobic bottles for routine blood cultures.|
|Body fluid||Cleanse surface with sterile saline or alcohol. Do not submit swabs.||Utilize anaerobic transport system for anaerobic cultures.||If inoculation of blood culture bottles with body fluid is performed by clinician, a separate sterile tube of fluid is required for direct Gram stain.|
|Catheter||Blood cultures may be drawn from blood catheters for assessment of line infections.||Sterile screw-cap container.||Do not submit urinary Foley catheters to the laboratory for culture.|
|Cerebrospinal fluid (CSF)||Disinfect area before collection.||Never refrigerate CSF.||Refrigeration prevents growth of some clinically significant organisms.|
|Decubitus ulcer||Cleanse surface. Obtain biopsy of base of ulcer or bone. Do not submit swabs.||Sterile tube for tissue for aerobic bacterial culture; anaerobic transport for anaerobic culture.||Due to high probability of colonizing microorganisms, swabs of decubitus ulcers should be rejected.|
|Ear||Collect fluid via aspiration or flexible swab, if needed.||As rapidly as possible; within 2 hours is optimal.||For otitis externa, vigorous swabbing of external surface is often necessary.|
|Eye||Moisten conjunctival swabs with sterile saline before collection.||As rapidly as possible; within 2 hours is optimal.||Clinicians who inoculate media themselves should work closely with the laboratory to ensure appropriate media are used.|
|Feces||Many different collection devices are available for ova and parasite examination of stool.||Cary–Blair is commonly used as a transport medium for culture and other studies.||Loose, not solid, stool should be collected for Clostridioides (Clostridium) difficile testing. Rectal swabs are inferior collection specimens compared with feces.|
|Genital||Swabs may be used for certain locations, such as the urethra.||As rapidly as possible; within 2 hours is optimal.||Refer to local laboratory guidelines for collection and transport needs for Chlamydia spp. and Neisseria gonorrhoeae testing.|
|Respiratory||Expectorated sputum should be collected after deep cough to avoid oral flora. Nasopharyngeal or throat swabs may be collected.||Swabs should be kept moist during transport.||Anterior nose cultures are reserved for detection of Staphylococcus carriers or visible nasal lesions. Endotracheal aspiration specimens are frequently contaminated with colonizing organisms and are suboptimal specimens to assess for respiratory infection.|
|Skin||Scrapings may be obtained for virus detection. Skin, hair, or nail collections may be obtained for detection of fungi.||Smears should be rapidly transported to the laboratory.||Smears may be performed by the clinician and rapidly transported to the laboratory for studies. Oral mucosal lesions may be tested for virus or other pathogens.|
|Tissue||Sterile saline should be added to keep pieces of tissue moist.||Utilize anaerobic transport system for anaerobic cultures.||Submit as much tissue as possible. Avoid submitting swabs that have been rubbed over the tissue surface.|
|Urine||First-void urine is required for Chlamydia and N. gonorrhoeae molecular testing. Midstream urine should be collected for bacterial culture.||Boric acid is commonly used as transport medium for culture and other studies.||Utility of urine cultures from indwelling catheters is limited.|
Specimens and Collection Devices
Although convenient for collection, swabs are suboptimal collection devices for culture for a variety of reasons. Most importantly, limited patient material is captured on a swab. If multiple culture-based or molecular assays are ordered from a single swab, insufficient material is available to distribute among the tests. Recently, flocked swabs have replaced traditional spun cotton swabs at many institutions due to the superior recovery of organisms compared with traditional nonflocked swabs. Flocked swabs are made from nylon fibers that are sprayed onto a shaft, allowing for optimal specimen absorption and release. Microorganisms are less likely to be caught within the fibers of a flocked swab compared with spun swabs. Regardless of whether flocked or nonflocked swabs are used, comparatively limited material is captured on a swab. Additionally, some swab types are toxic to specific organisms and will inhibit their growth in culture. For instance, calcium alginate swabs inhibit growth of Neisseria gonorrhoeae and some viruses. Wooden-shafted swabs are toxic to viruses and some bacteria. Swabs should not be submitted for anaerobic bacterial culture. Some molecular assays are inhibited with the use of certain swabs. When swabs must be used to collect very small amounts of fluid in limited spaces (e.g., collection of fluid from the inner or middle ear), they should not be allowed to dry. Swabs for respiratory virus testing may be polyester, rayon, or Dacron material with either plastic or aluminum shafts.
Medical devices or prosthetic material may be removed to assess for infection. Such devices are either sonicated or vortexed to remove the biofilm material for culture or other tests. Tissue biopsies must be maintained in a moist environment with sterile gauze moistened with sterile saline and placed in a sterile screw-capped container. Biopsies should be transported to the laboratory as rapidly as possible. In general, larger pieces of tissues are desirable. When multiple tests are desired, a piece or pieces of tissue approximately 1 to 2 cm 3 in size (i.e., about the size of one to two green peas) are required. When skin or deep tissue biopsies are collected, cleansing of the skin should be performed to avoid contamination with colonizing skin flora. When osteomyelitis is suspected, the bone itself should be collected rather than the overlying skin or subcutaneous tissue, after adequate cleansing of the overlying tissue.
For suspected bloodstream infections, blood cultures are critical. At a minimum, two blood cultures sets per order should be drawn from two different sites for adults, with each set composed of at least two blood culture bottles: one for isolation of aerobic pathogens and one for isolation of anaerobic organisms. The preferred blood volume per bottle for adult patients is 10 mL, for a minimum of 40 mL of blood drawn per blood culture order. Many factors are involved with optimal performance of blood culture testing, starting with complete disinfection of the skin surface to minimize contamination with normal human skin flora (e.g., coagulase-negative staphylococci, Corynebacterium spp., Bacillus spp. [not B. anthracis ], etc.). Generally, although a single blood culture bottle positive for one of these bacteria represents contamination, recovery of the same organism from multiple serially drawn blood cultures is more likely to represent clinically significant bacteremia. The majority of laboratories strive to maintain a contamination rate of <3%. The number of blood cultures sets drawn, the number and type (i.e.¸ aerobic vs. anaerobic) of blood culture bottles per set, and the amount of blood drawn per bottle also affect the efficiency of pathogen recovery from septic patients.
It is also imperative that proper collection containers are used for blood obtained for serologic, molecular, or other studies. Serum, plasma, whole blood, or other blood components may be required, depending on the type of testing performed. In blood, plasma is the liquid component of the blood—minus the white and red blood cells—before the blood has been allowed to clot and thus contains clotting factors. Serum is the liquid component of the blood minus clotting factors and cells. The type of anticoagulant also affects testing.
Select Important Considerations for Specimen Collection and Transport
Cerebrospinal fluid (CSF) collected for bacterial culture should never be refrigerated, because certain bacteria such as Neisseria meningitidis may not survive at low temperatures. Direct inoculation of synovial fluid and other body fluids into blood culture bottles has been shown to improve recovery of most organisms compared with direct plating of specimens in the laboratory onto solid agar media. This practice has also been applied to fluid other than joint fluids, such as peritoneal and pleural fluids. If body fluids are inoculated at the bedside by clinicians, a sterile tube of fluid should also be collected and submitted to the laboratory for a direct Gram stain.
Fresh urine collected without preservatives must be transported immediately to the laboratory within 30 minutes for culture; otherwise, urine may be stored in a sterile container at refrigerated temperatures for 24 hours before plating for culture. Urine may also be collected in boric acid preservative. Midstream (also known as clean-catch ) urine is recommended for bacterial culture and requires cleansing of mucosal or skin surfaces before collection. First-voided urine (i.e., voiding the first 20 to 30 mL of urine into a collection container) is recommended for detection of the sexually transmitted pathogens Chlamydia trachomatis , N. gonorrhoeae , and Trichomonas vaginalis . Collection of urine from urinary catheters for culture is discouraged due to biofilm production and colonization of the catheters, unless the collection occurs by straight catheterization rather than via an indwelling catheter. Urinary Foley catheters themselves should never be submitted for culture.
Sinus contents for bacterial culture should be collected by aspiration rather than by swabbing due to contaminating normal flora.
For assessment of Clostridioides (Clostridium) difficile , loose stool should be collected from patients with diarrhea (i.e., three or more loose stools within a 24-hour period). Formed stool should not be collected for C. difficile due to the likelihood of detecting colonizing C. difficile organisms.
Swabs should not be submitted for anaerobic culture, as the majority of anaerobes will not survive on the swab. Rather, such specimens should be collected in approved anaerobic transport vials, which often have indicators that monitor the maintenance of an anaerobic environment.
Samples for virologic culture and other studies are typically collected in a liquid transport medium, which protects viruses from dying. Swabs are also commonly used for molecular viral studies from certain body sites, such as the nasopharynx. Nasopharyngeal swabs are optimal collection devices for molecular testing for influenza, respiratory syncytial virus, and other respiratory viruses. Throat swabs are inferior for viral detection compared with nasopharyngeal swabs. Although sputum is also suboptimal for viral culture, it may be an acceptable source for molecular viral testing. Viral studies on blood may be performed using various blood fractions (e.g., serum, plasma, or whole blood). Specimens for viral culture studies must not be stored at −20° C due to the freeze–thaw cycling of such freezers, which damages the organisms.
Transport of specimens for fungal studies is best achieved at room temperature. However, if Mucorales (also referred to as Zygomycetes ) infection is clinically suspected, rapid transport of the specimen to the laboratory is necessary for adequate recovery in culture due to the fastidious nature of such organisms. In addition, crushing of tissue with a grinder might damage the Mucorales hyphae. Because most laboratories grind tissue to break apart the specimen for easier plating across various media, clinicians should notify laboratories when Mucorales infection is suspected. If notified, laboratories will often mince the tissue into small pieces with a sharp blade before plating, rather than grinding or crushing. CSF for fungal culture should be held at room temperature before plating.
Specific collection tubes or bottles for mycobacterial blood cultures may be utilized in some laboratories. Gastric aspirates collected for mycobacteria should be transported immediately to the laboratory because acid-fast bacilli may not survive the high acidity environment of the stomach.
Blood smears for examination of parasites should be prepared optimally within 1 hour of collection for best morphology. Thick and thin smears allow for examination of the parasitemia percentage, as well as species-level identification of Plasmodium . Stool specimens for ova and parasite (O&P) examination may be performed on fresh stool or stool in appropriate preservatives. EIAs and molecular studies may also be performed on stool. Various enema preparations may interfere with stool O&P or other parasitologic stool assays; therefore specimen collection may need to be delayed for a period after enemas.
Diagnostic Methods in the Clinical Microbiology Laboratory
The clinical microbiology laboratory has become an incredibly diverse space with respect to the diagnostic and confirmatory testing methods that are offered and maintained for routine patient care. These methods range from traditional stain and culture-based techniques for bacterial, mycobacterial, fungal, viral, and parasitic pathogens to increasingly complex nucleic acid amplification tests (NAATs) (e.g., single-target assays vs. syndromic, multitarget panels), whole-genome sequencing (WGS) and next-generation sequencing (NGS) assays for direct-from-specimen pathogen detection, strain typing, and identification of antimicrobial resistance markers. It is important that clinicians be aware of how these methods generally work, including their advantages and limitations, so that results are appropriately interpreted. This section will provide brief overviews on select, commonly performed testing methodologies in clinical microbiology laboratories.
Many different stains are used in the microbiology laboratory for the detection and visualization of microorganisms directly from patient specimens and/or after growth in culture; however, only those most commonly used will be reviewed here ( Table 2.2 ). Stains can be classified as either “simple” contrast stains, differential stains, or fluorescent stains. Contrast stains contain charged dyes that are either attracted to or repelled from microorganisms. This includes lactophenol blue, which nonspecifically stains fungal cell walls, and the classic India ink stain for detection of Cryptococcus neoformans/gattii in CSF. In contrast, differential stains require more than one stain, have multiple steps (i.e., stain, decolorize, counterstain), and allow for a distinction between cellular morphology and organization. The most widely used differential stains include the Gram stain for bacteria, acid-fast stains for detection of Mycobacterium spp., and modified acid-fast stains for visualization of select members of the aerobic actinomycetes (e.g., Nocardia spp.) and certain intestinal parasites (e.g., Cyclospora spp., Cryptosporidium spp., Cystoisospora ) (see Table 2.2 ). Briefly, Gram-positive cells (bacteria and yeast) stain bluish/purple due to intercalation of the crystal violet dye within the thick peptidoglycan layer of the cell wall and retain the stain after decolorization using a mild alcohol ( Fig. 2.1 ). In contrast, although Gram-negative cells also have cell wall peptidoglycan, it is significantly less abundant and cannot retain the crystal violet after decolorization. Gram-negative cells appear reddish-pink due to retention of the safranin counterstain (see Fig. 2.1 ). Importantly, the Gram stain is not useful for detecting very thin or small bacteria (e.g., Treponema spp., Borrelia spp., etc.), bacteria without a cell wall (e.g., Mycoplasma spp., Ureaplasma spp.), and Mycobacterium spp. Acid-fast stains are required for members of the Mycobacterium genus due to the thick, hydrophobic layer of mycolic acid in the cell wall of these organisms. Multiple different acid-fast staining procedures have been developed, primarily differing in the method used to penetrate the thick lipid layer, either by heating the slides using the Ziehl–Neelsen method or using a higher concentration of the primary carbolfuchsin stain, which has a lipid-soluble phenol group allowing the stain to penetrate the cell wall without heat (see Table 2.2 ). After the primary stain, slides are decolorized with an acid–alcohol and counterstained with methylene blue. Resistance to decolorization by the acid–alcohol after carbolfuchsin staining (i.e., “acid-fast”) is required for an organism to be termed “acid-fast” and will appear pinkish-red. Some organisms are partially acid-fast (e.g., Nocardia spp., Cryptosporidium spp., Cyclospora spp., etc.), meaning that they do not stain evenly with carbolfuchsin using standard acid-fast staining methods. Modified acid-fast stains utilize milder decolorization acids for better visualization of such organisms (see Table 2.2 ). Because most laboratories do not routinely perform modified acid-fast stains on direct patient specimens, clinicians should notify the laboratory when nocardiosis is on the differential. Additional differential stains, their application, and “key laboratory pearls” are listed in Table 2.2 .
|Primary Application||Key Laboratory Pearls|
|India ink||Cryptococcus spp.|
|Lactophenol aniline blue||Fungi|
|Gram stain (GS)||Bacteria, yeast|
|Acid-fast (e.g., Ziehl–Neelsen, Kinyoun, modified Kinyoun stains)||Mycobacterium , aerobic actinomycetes, select parasites|
|Trichrome, modified trichrome, iron–hematoxylin||Intestinal protozoan trophozoites/cysts, microsporidia|
|Giemsa and Wright–Giemsa||Parasites in blood|
|Calcofluor white||Fungi, select parasites|
|Direct fluorescent antibodies||Varies|