SPECIMEN PROCESSING GRAM STAINING -one of the most valuable procedures performed by the microbiology laboratory. -rapidly provides information that is used by the clinician for selecting appropriate antimicrobial therapy -To prepare a smear for staining, an aliquot of the most purulent or bloody portion of the specimen is placed on a clean microscopic slide in a manner that provides both thick and thin areas. For sterile body fluids, a cytocentrifuge may be used to concentrate the specimen by 10–100 times (Peterson, 1988). The material on the slide is allowed to air-dry, is fixed with methanol or gentle heat, and then is stained with Gram stain reagents (crystal violet, Gram iodine, alcohol, and safranin). Organisms that have a gram-positive cell wall will resist decolorization with methanol and will retain the purple color of the crystal violet; organisms that have a gram- negative cell wall will be decolorized and will stain red with safranin counterstain. The organisms observed should be evaluated for size, shape, and Gram reaction, which should be reported with as much description as possible; CULTURE TECHNIQUES -Media for culture are selected to provide the optimal conditions for growth of pathogens commonly encountered at a particular site or in a particular type of specimen -the media chosen may include selective and differential media, in addition to standard enrichment agar. -Blood-supplemented agar is a good general growth medium and can be used to demonstrate the hemolytic action of colonies on the red blood cells Antibiotics or chemicals can be added to create a selective medium such as colistin–nalidixic acid (CNA) agar or phenylethyl alcohol agar, both of which are used to inhibit the growth of gram-negative bacilli while permitting gram-positive bacteria to grow -Heating the blood to make chocolate agar and adding vitamin supplements creates an enriched medium with available hemin (X factor) and nicotinamide adenine dinu- cleotide (V factor) for the isolation of Haemophilus spp. and other fastidious bacteria. -Gram-negative bacilli may be separated from gram-positive bacilli by using bile salts and dye in a medium such as MacConkey’s agar, which additionally divides the colonies into lactose-positive and lactose- negative colonies, thus making it both selective and differential. -Bacterial cultures are generally incubated at 35° C and are examined initially after 18–24 hours of incubation. -Addition of 5%–10% carbon dioxide (CO2) may be essential or stimulatory to the growth of N. gonorrhoeae, Haemophilus influenzae, and S. pneumoniae, and should be used whenever feasible. -For recovery of anaerobes, inoculated media should be placed into an anaerobic environment as quickly as possible. Several types of anaerobic culture systems are available. One of these is the anaerobic jar, in which water is added to a CO 2 and hydrogen (H2) generator package, and oxygen (O2) is catalytically converted to water with palladium-coated alumina pellets contained in a lid chamber A modification of this system is a transparent plastic bag containing its own gas generator and palladium catalyst, and designed to hold an agar plate; these are often referred to as anaerobic Bio-Bags. Another approach to anaerobic culture is the anaerobic glove box or chamber, which consists of a large, clear plastic, airtight chamber filled with an oxygen-free gas mixture of nitrogen, hydrogen, and carbon dioxide. Specimens, plates, and tubes are introduced into or removed from the chamber through a gas interchange lock. Anaerobiosis is maintained by palladium catalysts and the hydrogen gas in the chamber. All manipulations within the chamber are done with neoprene gloves sealed to the chamber wall or, for “gloveless” systems, through a hole with sleeves that seal tightly around the forearms. The chambers contain internal incubators that main- tain the incubation temperature. Each of the anaerobe systems has its advantages and disadvantages, but all are equally effective in isolating clinically significant anaerobic bacteria from specimens. A system for processing anaerobic specimens under a constant anaerobic environnent to reduce the chance of excess exposure to oxygen is available in the Anox- amat system (Summanen, 1999). -Bacterial cultures should be examined routinely after 18–24 hours of incubation. -The exception to this is the anaerobe culture, which is gener- ally examined at 48 hours to allow these slower growing bacteria to produce visible colonies. -In general, solid media are held for 48 hours, with liquid media held for an additional 24–48 hours. -A preliminary report is issued when the culture is first examined; this report is updated as additional information becomes available. Certain results (e.g., positive blood or CSF Gram stain, isolation of an organism requiring infection control measures) are reported to the healthcare pro- vider as soon as the information becomes available. Final reports are issued when all work on a culture has been completed. GENERAL PRINCIPLES OF SPECIMEN COLLECTION AND HANDLING TIMING OF SPECIMEN COLLECTION For optimal detection of the pathogens responsible for an infectious disease, specimens should be collected at a time when the likelihood of recovering the suspected agent is greatest -Specimens for recovery of bacteria should ideally be collected before anti- microbial therapy is started. SPECIMEN VOLUME The volume of specimen collected must be adequate for performance of the microbiological studies requested. SPECIMEN COLLECTION Specimens should be obtained from the site of infection with minimal contamination from adjacent tissues and organ secretions and, with the exception of stool, should be collected in a sterile container. All specimens should be labeled with the name and identification number of the person from whom the specimen was collected, the source of the specimen, and the date and time it was collected. SPECIMEN TRANSPORT After collection, specimens should be placed in a biohazard bag and transported to the laboratory as soon as possible. If a delay is unavoidable urine, sputum and other respiratory specimens, stool, and specimens for detection of C. trachomatis or viruses should be refrigerated to prevent overgrowth of normal flora. Cerebrospinal fluid (CSF) and other body fluids, blood, and specimens collected for recovery of N. gonorrhoeae should be held at room temperature, because refrigeration adversely affects recovery of potential pathogens from these sources. UNACCEPTABLE SPECIMENS • Any specimen received in formalin • 24-hour sputum collections • Specimens in containers from which the sample has leaked • Specimens that have been inoculated onto agar plates that have dried out or are outdated • Specimens contaminated with barium, chemical dyes, or oily chemicals • Foley catheter tips • Duplicate specimens (except blood cultures) received in a 24-hour period • Blood catheter tips submitted for patients without concomitant positive blood culture The following specimens should be rejected for anaerobic culture: • Gastric washings • Urine other than suprapubic aspirate • Stool (except for recovery of Clostridium difficile for epidemiologic studies or for diagnosis of bacteria associated with food poisoning) • Oropharyngeal specimens, except deep tissue samples obtained during a surgical procedure • Sputum • Swabs of ileostomy or colostomy sites • Superficial skin specimens UNIVERSAL PRECAUTIONS Universal precautions must be followed when handling all specimens. Appropriate barriers are used to prevent exposure of skin and mucous membranes to the specimen. Gloves and a lab coat must be worn at all times, and masks, goggles (or working behind a plastic shield), and imper- meable gowns or aprons must be worn when there is risk of splashes or droplet formation. -Optimally, all specimen containers but, at a minimum, those containing respiratory secretions and those submitted specifically for detection of mycobacteria or fungi should be opened in a biological safety cabinet. BLOOD Note: Culture of blood is essential in identifying bacteria responsible for bacteremia, sepsis, infections of native and prosthetic valves, suppurative thrombophlebitis, mycotic aneurysms, and infections of vascular grafts. -In general, blood should be collected for culture before beginning antimicrobial therapy when any one or a combination of the following are present: fever (38° C or greater), hypothermia (36° C or lower), leukocytosis (especially with a left shift), granulocytopenia, or hypotension. SPECIMEN COLLECTION Note: Timely detection and accurate identification of organisms in the blood depend on appropriate collection, transport, and processing of the specimen. -To minimize contamination of blood specimens by skin flora, the venipuncture site should be prepared with a bactericidal agent. -The skin first is cleaned with alcohol (70% isopropyl or ethyl alcohol) and then with a 1–2% iodine solution, an iodophor, or chlorhexidine. -For maximum antisepsis, the area should dry for 1–2 minutes before venipuncture of the selected peripheral vein. APPROPRIATE TIMING FOR DETECTION OF BACTEREMIA AND FUNGEMIA -The optimal time to draw blood for cultures when bacteremia or fungemia is suspected is just before a chill but, because this is not predictable, most blood cultures are collected after the onset of fever and chills. -Blood is drawn with a needle and syringe and, without changing needles, is injected directly into bottles of culture media or other blood culture system -The inoculated bottles are immediately inverted several times to ensure mixing, and then they are transported to the laboratory at room temperature as soon as possible after collection. Blood cultures should never be refrigerated. SPECIMEN VOLUME Note: In adults with bacteremia, the number of colony-forming units (CFU) per milliliter of blood is frequently low. Therefore, for adults, collecting 20– 30 mL of blood per culture set is strongly recommended In infants and children, the concentration of microorganisms in blood is higher, and collection of 1–5 mL of blood per culture is adequate. Recommendations concerning the number of blood specimens to collect are based on the nature of the bacteremia: transient, intermittent, or continuous. Transient bacteremia follows manipulation of a focus of infection (e.g., an abscess, a furuncle, or cellulitis), instrumentation of a contami- nated mucosal surface (as occurs during dental procedures, cystoscopy, urethral catheterization, suction abortion, or sigmoidoscopy), or a surgical procedure in a contaminated site (e.g., transurethral resection of the pros- tate, vaginal hysterectomy, colon resection, and debridement of infected burns). It ccurs early in the course of many sys- temic and localized infections such as meningitis, pneumonia, pyogenic arthritis, and osteomyelitis two or three 20-mL blood samples drawn over a 24-hour period and equally distributed into aerobic and anaerobic blood culture bottles is suf- ficient to detect most bloodstream infections. One investigator demon- strated that a total of four blood cultures drawn within 24 hours increased the yield of potential pathogens by as much as 20% The optimal time interval between cultures is unknown, but 30–60 minutes for the first two sets has been suggested, with another one to two sets drawn over the remaining 24 hours if symptoms of septicemia persist However, if initiation of antimicrobial therapy is deemed urgent, cultures should be collected before therapy is begun, from separate sites within a few minutes. RECOVERY OF MICROORGANISMS Host factors such as antibodies, complement, phagocytic white blood cells, and antimicrobial agents may impede recovery of microorganisms from blood; therefore, various approaches have been used to counteract these factors. -Diluting the blood specimen in broth medium in a 1:10 ratio provides optimal neutralization of the serum bactericidal activity -Incorporating 0.02–0.05% sodium polyanethol sulfonate in the blood culture medium inhibits coagulation, phagocytosis, and complement activation, and inactivates aminoglycosides. BLOOD CULTURE SYSTEMS Automated detection systems are rapidly replacing manual systems. Both automated and manual systems use nutritionally enriched liquid media, which are capable of supporting growth of most bacteria. Traditionally, two bottles are inoculated, and one is vented to ensure recovery of aerobes. -Routine inoculation of two aerobic media and only selective use of anaerobic blood cultures may allow detection of more bacteremias and fungemias. 1. Manual Blood Cultures Two commercial manual blood culture systems are available. -biphasic system consist of a broth medium in a bottle to which a chamber containing agar media on a paddle is attached. To subculture this system, the bottle is tipped, allowing the blood–broth mixture to enter the chamber and flow over the agar media. Colonies on the agar medium are used for identification and susceptibility testing For recovery of aerobic and facultative bacteria and yeasts, this system is comparable to or better than other systems -lysis–centrifugation blood culture system possible. Blood is added to the tube, which is inverted several times to prevent clotting and transported to the laboratory as soon as Ideally, the specimen is processed immediately, but processing can be delayed for up to 8 hours without adversely affecting recovery of micro- organisms. To process the culture, the tube is centrifuged for 30 minutes at 3000 g, the supernatant is discarded, and the sediment is mixed on a vortex mixer and plated onto agar media Advantages of lysis–centrifugation include excellent recovery of Staphylococcus aureus, some Enterobacteriaceae, and fungi (it is the best system for recovery of H. capsulatum), the direct availability of colonies for identification and susceptibility testing, and the ability to carry out quantitative cultures. Moreover, this system is flexible because special media can be inoculated to recover organisms with specific growth requirements, such as species of Legionella and mycobacteria. However, the system is labor-intensive, is less likely to recover Streptococcus pneumoniae, Haemophilus influenzae or anaer- obes, and the risk for contamination is increase 2. Automated Blood Cultures -much less labor-intensive than the “manual” systems -the usual incubation period can be shortened from 7 to 5 days (1)-One such system is based on the colorimetric detection of carbon dioxide (CO2) produced during microbial growth (Thorpe, 1990). A CO2 sensor is bonded to the bottom of each blood culture bottle and is separated from the broth medium by a mem- brane that is impermeable to most ions and to components of media and blood but freely permeable to CO 2 bottles. Inoculated bottles are placed in cells in the instrument, which provides continuous rocking of both aerobic and anaerobic If bacteria are present, they generate CO2, which is released into the broth medium; the pH then decreases, causing the sensor to change color from green to yellow. Color changes are monitored once every 10 minutes by a colorimetric detector. Media available for use with this system include routine aerobic and anaerobic media, which accommodate 5–10 mL of blood; Pedi-BacT, which accommodates 4 mL of blood or less; and fastidious antibiotic neutralization, which enhances recovery of fungi and recovery of bacteria from patients receiving anti- microbial agents. (2) A second continuous-monitoring system is based on fluorescent technology Bonded to the base of each vial is a CO2 sensor that is impermeable to ions, medium components, and blood but freely perme- able to CO2. If organisms are present, they release CO2 into the medium it then diffuses into the sensor matrix and generates hydrogen ions The subsequent decrease in pH increases the fluorescence output of the sensor, changing the signal transmitted to the optical and electronic components of the instrument. The computer generates growth curves, and data are analyzed according to growth algorithms. Inoculated bottles are placed in individual cells of the instrument, in which both aerobic and anaerobic bottles are continuously rocked. Aerobic and anaerobic low-volume (5–7 mL of blood) and high-volume (8 to 10 mL of blood) media, Peds- Plus medium (0.5 to 5 mL of blood), and the Myco F Lytic medium for recovery of fungi and mycobacteria are available (Waite, 1998). (3) A third system detects growth of organisms in broth by measuring gas consumption and/or gas production Each inoculated vial is fitted with a disposable connector that contains a recessed needle. needle penetrates the bottle stopper and connects the bottle headspace to the sensor probe. The sensor monitors changes within the headspace in the consumption and/or production of all gases (CO 2, N2, and H2) by growing organisms and creates data points internally in the computer. Two basic types of medium are available: aerobic and anaerobic media that contain 80 mL of broth and accommodate 0.1–10 mL of blood, and EZ . Draw (direct draw) aerobic and anaerobic bottles that contain 40 mL of broth and accommodate 0.1–5 mL of blood. DETECTION OF RARELY ENCOUNTERED BACTERIA -Detection of some bacteria requires prolonged incubation or special media. -For example, when brucellosis is suspected, blood should be collected early in the disease and cultures should be incubated for 2– 3 weeks, although with the automated systems brucellas often grow in less than 1 week - Infections with species of Borrelia, except Borrelia burgdorferi (the etiologic agent of Lyme disease, most commonly diagnosed serologically), are diagnosed by detecting spirochetes in the peripheral blood during febrile periods. - To isolate Leptospira interrogans from blood, a few drops of fresh or anticoagulated blood collected during the first week of illness are added to each of three to four tubes of leptospiral semisolid culture medium (Fletcher’s medium or Ellinghausen–McCollough–Johnson– Harris medium). - Two methods may be used to recover mycobacteria from blood specimens. With the lysis–centrifugation technique, (1) a concentrate is pre- pared, (2) the sediment is inoculated to solid and/or liquid media, and (3) the cultures are incubated for up to 8 weeks. more rapid approach is direct inoculation of the liquid medium developed by the manufacturer of automated and semiautomated broth culture systems specifically for recovery of mycobacteria. DETECTION AND NOTIFICATION OF POSITIVE CULTURES - Positive blood cultures containing commonly isolated aerobic organisms are usually detected within 12–36 hours of incubation - The initial report is a Gram stain report only. Identification and susceptibility results can be expected within 24–48 hours after the Gram stain report. - Cultures containing anaerobes are usually not detected for 48–72 hours, and identification is not available for 3–4days after that - Fastidious organisms, such as those found in the HACEK group (Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella) may not be detected until 3–5 days. BODY FLUIDS CEREBROSPINAL FLUID CSF is collected to diagnose meningitis and, less frequently, viral encepha- litis. Potential pathogens are listed in Table 63-1. TABLE 63-1 Infectious Meningitis Syndromes Syndrome Onset/Duration Probable Pathogens Acute Subacute Chronic <24 hours 1–7 days Persisting at least 4 weeks Pyogenic bacteria Enteroviruses, pyogenic bacteria Mycobacterium tuberculosis Treponema pallidum Brucella sp. Leptospira interrogans Borrelia burgdorferi Cryptococcus neoformans Coccidioides immitis Histoplasma capsulatum Sample Collection and Transport - As when collecting blood for culture, careful skin antisepsis is essential for collection of CSF, which typically is submitted to the laboratory in three or occasionally four tubes. - Suggestions for tests performed on fluid in each tube are as follows: tube 1, protein and glucose; tube 2, preparation of smears to stain with the Gram stain or other stains and for culture; tube 3, cell counts; and, if indicated, tube 4, special tests such as the cryptococcal antigen, serologic test for syphilis, molecular tests or other serologic studies, and cytology. - CSF should be transported promptly to the laboratory and processed as rapidly as possible. - If a brief delay in processing is unavoidable, the specimen should be held at room temperature unless viral culture is requested, in which case a portion (preferably 1 mL but no less than 0.5 mL) may be refrigerated for a short time. - Specimen processing differs for bacteria, fungi, viruses, and parasites Sample Processing for Bacterial and Fungal Culture - Processing CSF for routine bacterial culture includes concentration (if 1 mL or more of specimen is received), preparation of a smear by cyto- centrifugation for staining with Gram stain, and culture. Concentrate the fluid by centrifugation at a minimum of 1500 g for 15 minutes. The supernatant is decanted into a sterile tube, leaving about 0.5 mL of sediment and fluid, which is thoroughly mixed on a vortex mixer or by forcefully aspirating up and down into a sterile pipette. Diagnosis of chronic bacterial meningitis requires specific requests because the CSF is handled differently for each entity. - To diagnose brucellosis, the CSF is processed as described earlier for routine bacterial culture, but the media are incubated for 2–3 weeks. -- For leptospirosis, Leptospira interrogans may be cultured from the CSF during the first few weeks of illness. Special media (listed earlier under blood) are inoculated with a few drops of CSF and incubated -Processing CSF for detection of mycobacteria is indicated only for samples with pleocytosis, or decreased glucose, or elevated protein values For optimal recovery, culture of at least 5 mL is recommended. The fluid is centrifuged at 3000–3600 g for 30 minutes, the supernatant is decanted, and the sediment is thoroughly mixed on a vortex mixer and used to prepare smears for staining and to inoculate appropriate media Nucleic acid amplification, using a modification of a commercial assay or an assay developed in-house and validated, may be useful for direct detection of Mycobacterium tuberculosis complex in CSF Additional Diagnostic Tests In addition to smears stained with the Gram stain and bacterial culture, the supernatant of a centrifuged specimen or the original fluid may be used to perform latex agglutination tests for detection of antigens of Streptococcus agalactiae, S. pneumoniae, some serotypes of Neisseria meningitidis, Esche- richia coli (the K1 capsular antigen cross-reacts with that of N. meningitidis type B), and H. influenzae type b. These latex tests are most useful in diagnosing partially treated meningitis (Bhisitkul, 1994; Maxson, 1994) and in confirming a positive Gram-stained smear. The routine use of latex tests should be discouraged because, compared with smears stained with Gram stain, their sensitivity is not significantly greater and they are much more expensive (Kiska, 1995; Perkins, 1995). Although the value of latex tests for diagnosis of bacterial meningitis is questionable, an immu- nochromatographic test that detects the C polysaccharide cell wall antigen common to all serotypes of S. pneumoniae, has been shown to be very useful for rapid diagnosis of pneumococal meningitis when testing CSF (Werno, 2008). OTHER BODY FLUIDS Fluid is collected from the pericardial, thoracic, or peritoneal cavity, or from joint spaces, by aspirating with a needle and syringe. - A volume of 1–5 mL is adequate for isolating most bacteria, - 10–15 mL is optimal for recovery of mycobacteria and fungi, which are generally present in low numbers. Moreover, to diagnose peritonitis associated with chronic ambulatory peritoneal dialysis, collection of at least 50 mL of fluid may improve recovery of the responsible pathogen - To transport the fluid, it is aspirated into a sterile container and delivered promptly to the laboratory. Alternatively, peritoneal fluid may be directly inoculated into blood culture bottles at the patient’s bedside; however submission of fluid in blood culture bottles eliminates the possibility of direct Gram staining and delays the identification and susceptibility testing of any pathogens isolated. Sample Processing for Bacterial Culture Processing fluid from body cavities for detection of bacteria involves preparing a smear for Gram stain and inoculating appropriate media for culture. the sample may be inoculated into blood culture bottles at the bedside, although this is not optimal, or it may be processed in the laboratory. -In the laboratory, the fluid is centrifuged at 1500–2500 g for 20–30 minutes. The supernatant is removed, leaving about 0.5 mL fluid in addition to the sediment, which is mixed thoroughly and then used to prepare smears and inoculate media. Alternatively, a small volume of noncloudy, nonviscous fluid (about 0.1 mL) may be removed before centrifugation and used to prepare a cytocentrifuged smear. resPiraTory TracT NASOPHARYNGEAL SPECIMENS Nasopharyngeal aspirates, washings, and swab specimens are collected predominantly for diagnosis of viral respiratory infections but also pneumonia due to C. trachomatis or Chlamydophila pneumoniae, pertussis, and rarely diphtheria. Specimens from the nose also are used to identify carriers of methicillin-resistant S. aureus (MRSA). Specimen Collection, Transport, and Processing -Nasopharyngeal aspirates and washings are superior to swabs for recovery of viruses, but swabs frequently are submitted because they are more convenient. - Washings or swab specimens are collected for detection of Bordetella pertussis; - a swab is the preferred specimen for C. trachomatis, C. pneumoniae, and Corynebacterium diphtheriae. - An aspirate is collected with a plastic tube (e.g., one used to feed premature infants) attached to a 1-mL syringe or a suction catheter with a mucus trap - A wash is obtained with a rubber suction bulb by instilling and withdrawing 3–7 mL of sterile phosphate-buffered saline. - To collect nasopharyngeal cells with a swab, all mucus from the nasal cavity is removed, then a small flexible nasopharyn- geal swab is inserted along the nasal septum to the posterior pharynx and rotated against the mucosa several times. - For detection of C. trachomatis, a nasopharyngeal swab specimen is collected with a polyester-tipped swab, which may be used for culture or for preparation of a smear for direct fluorescent antibody staining. - To detect B. pertussis by culture, inoculation of washings or swab speci- mens (preferably collected with a calcium alginate swab) at the bedside is optimal. If this is not possible, the sample is placed into sterile casamino broth, transported promptly to the laboratory, and processed within 1–2 hours for culture If the sample must be sent to a reference laboratory for culture, the swab should be inoculated into and left in a solid transport media such as Regan–Lowe or Jones–Kendrick, incubated at 37° C for 48 hours, and then shipped at ambient temperature However, the most sensitive method for detecting B. pertussis is PCR, for which the optimal sample is a nasopha- ryngeal swab specimen collected with a Dacron swab (a-lginate fibers and aluminum in the shaft are inhibitory). THROAT SPECIMENS - most commonly collected to diagnose group A streptococcal pharyngitis; - Throat swabs received in the clinical laboratory for routine bacterial culture should be evaluated for these streptococci only. Throat washings or swab specimens are useful for detection of viruses shed in oral secretions without causing pharyngitis (HSV, CMV, or enteroviruses). -Throat swab specimens may also be helpful in determining the etiologic agent of epiglottitis, a rapidly progressing cellulitis with the potential to cause obstruction of the airway (almost always due to H. influenzae type b, but occasionally S. aureus or S. pneu- moniae) and in diagnosing gonorrhea, Mycoplasma pneumoniae pneumonia, diphtheria, and Vincent’s angina. Specimen Collection and Transport - Throat swab specimens are collected by depressing the tongue with a tongue blade, introducing the swab between the tonsillar pillars and behind the uvula without touching the lateral walls of the buccal cavity, and swabbing back and forth across the posterior pharynx. Swab specimens collected for detection of viruses should be placed in a viral transport medium and those for detection of bacteria, in a tube transport system containing modified Stuart’s medium. Throat washings for diagnosis of viral infections are obtained by gargling with 5 mL of viral transport medium containing antibiotics. Throat washings and swab specimens should be delivered promptly to the laboratory, or refrigerated for a short time if a delay in transport is unavoidable. Specimen Processing For diagnosis of group A streptococcal pharyngitis culture is most sensi- tive; however, this requires overnight incubation Use of a selective medium increases the recovery of S. pyogenes by inhibiting overgrowth of normal flora; but because the amount of growth of S. pyogenes on selective media at 24 hours often is insufficient for testing, plates may need to be reincubated an additional day before confirmatory testing can be per- formed. For rapid diagnosis, a commercial direct probe is the most sensi- tive and can be used as a replacement for culture Other rapid, direct tests for group A streptococcus (several are commercially available) are less sensitive (as low as 70%); therefore, if one of these is used two throat swab specimens should be collected f the direct test is positive, the second swab may be discarded; but in children if the direct test is negative, confirmatory culture must be performed, using the second swab In adults, because the incidence of streptococcal infec- tion and the risk of rheumatic fever are low, diagnosis of group A strepto- coccal pharyngitis on the basis of a rapid, direct test, without confirming a negative result with culture, is an acceptable alternative To detect N. gonorrhoeae in the throat the swab specimen should be inoculated at the bedside or transported to the laboratory within 2 hours and inoculated as soon as possible on to a selective medium, such as modified Thayer–Martin agar. If a delay in processing is unavoidable, the swab should be held at room temperature. For diagnosis of diphtheria both nasopharyngeal and throat swab specimens are collected and transported to the laboratory immediately. If laboratory personnel are not experienced in the recovery and identification of C. diphtheriae, the specimens should be sent in a semisolid transport media (e.g., Amies) to a reference laboratory. A differential inhibitory medium containing potassium tellurite, such as Tinsdale medium, is optimal for cultivating C. diphtheriae. = This medium, however, is expensive, has a short shelf-life, and is difficult to obtain from commercial vendors; therefore it is seldom used in clinical laboratories. , but because CNA is not a differential medium, all diphtheroid colony types must be evaluated to exclude C. diphtheriae when this agent is suspected. In addition, a sheep blood agar plate should be inoculated and examined for group A streptococci. SPUTUM AND TRACHEAL ASPIRATES Microbiological studies of sputum (expectorated and induced) and tracheal aspirate specimens are done primarily to determine the etiologic agents of pneumonia. Tracheal aspirates represent lower respiratory secretions collected in a Lukens trap from patients with tracheostomies. Patients with tracheostomies rapidly become colonized with gram-negative bacteria and other potential nosocomial pathogens and because bacteria colonizing the respiratory tract cannot be differentiated from bacteria causing invasive disease by culture of tracheal aspirates, interpretation of routine culture results is difficult Specimen Collection and Transport -Optimally, expectorated sputum is collected early in the morning before eating. - The individual rinses his or her mouth with water and then expectorates a specimen, preferably 5–10 mL, resulting from a deep cough - For persons with nonproductive coughs, a specimen may be induced by allowing the individual to breathe aerosolized droplets of a solution of 15% sodium chloride and 10% glycerin for about 10 minutes or until a cough reflex is initiated. - Sputum and tracheal aspirate specimens should be delivered promptly to the laboratory or refrigerated for a short time if a delay is unavoidable. Specimen Processing - Both specimen types should be screened before they are plated for routine bacterial culture to determine whether they are representative of lower respiratory secretions or of saliva. A smear prepared from a portion of the specimen consisting of purulent material is stained with the Gram stain. In general, specimens with more than 10 epithelial cells per low-power field by screen (Fig. 63-2) are considered to have significant contamination with saliva and should be rejected. Specimens with fewer than 25 epithelial cells and more than 25 neutrophils per low-power field are probably acceptable (Murray, 1975). The number of neutrophils is not usually considered when determining specimen quality, because the individual from whom the sputum was collected may be neutropenic. Screening induced sputum specimens and expectorated sputum samples submitted for detection of M. pneumoniae, species of Legionella, and mycobacteria to assess their quality is not generally required . However, data from one study suggest that screening sputum specimens for the presence of neutrophils is an effective method to evaluate the acceptability of sputum for mycobacterial smear and culture (McCarter, 1996). - The Gram-stained smears prepared from specimens that are acceptable for culture are examined under oil immersion to determine the relative amounts of organisms. the quantity of organisms (rare, few, moderate, or many) is estimated for each kind of bacterium (e.g., gram-positive cocci in pairs (Fig. 63-3), chains or clusters; gram-positive bacilli; gram-negative diplococci, and gram-negative rods), noting whether or not they are intra- cellular. - Tracheal aspirates for which no organisms are observed in the Gram-stained smear should probably be rejected For specimens from persons with cystic fibrosis, also inoculating a medium selective for Burkholderia cepacia is recommended. When legionnaires’ disease is suspected, Legionella culture and a rapid, direct test (fluorescent antibody on a respiratory specimen or Legionella antigen on a urine specimen) are recommended. Direct fluorescent anti- body staining, which can provide results in several hours rather than the 3–7 days required for culture, should be used to supplement but not replace culture. PCR also can be performed but is considerably more expensive than culture, direct fluorescent antibody (DFA), or urine antigen testing. Culture is the most sensitive of these methods and should always be performed. Several drops of the specimen should be inoculated to each selective and nonselective buffered charcoal yeast extract agar plate. Use of the selective agar inhibits the growth of most other respiratory flora; however, some strains of Legionella are susceptible to the medium’s inhibi- tory agents. Thus a nonselective plate should always be included. -For optimal detection of mycobacteria in sputum, collection of three samples on three separate days is recommended. Sputum and other respiratory secretions must be decontaminated to prevent the normal respira- tory flora from overgrowing the slower-growing mycobacteria. This process and detection methods are discussed in Chapter 60. All specimens submitted for mycobacterial stain, culture, and molecular testing should be processed in a biological safety cabinet, preferably in an isolated room with negative air pressure (level 3 laboratory). BRONCHOSCOPY SPECIMENS Bronchoalveolar lavage fluid and protected brush specimens are useful for diagnosis of bacterial pneumonia in ventilated patients who have not received antimicrobial therapy, and for detection of opportunistic pathogens in immunocompromised patients with pneumonia ll bronchoscopy specimens should be plated quantita- tively to determine significance of potential pathogens recovered. Only protected brush specimens are suitable for anaerobic culture (Baselski, 1994). For culture of species of Legionella, sputum specimens are preferable because bronchoalveolar lavage samples are diluted with saline and may contain small amounts of the anesthetic used locally, which inhibits the organism. Specimen Collection and Transport -The protected brush sample is collected with a small brush that holds 0.001–0.01 mL of secretions, placed in a catheter, within a double cannula. The outer cannula has a displaceable polyethylene glycol plug at the tip To obtain a specimen, the cannula is inserted to the desired area via bronchoscopy, the inner cannula is pushed out, dislodging the protective plug (water-soluble), and the brush is extended even farther, beyond the inner cannula. Once the sample is taken, the brush is pulled back into the inner cannula, and both brush and inner cannula are pulled into the outer cannula to prevent contamination of the brush when the catheter is removed. The brush then is placed in 1 mL of sterile saline or broth. The specimen should be transported immediately to the laboratory and pro- cessed as soon as possible. If a delay is unavoidable, the specimen should be stored in the refrigerator. To collect bronchoalveolar fluid, the tip of the bronchoscope is care- fully wedged into an airway lumen. A volume of saline (usually >140 mL) in three to four aliquots is injected through the lumen, sampling an esti- mated 1 million alveoli. The total volume returned varies based on the volume instilled, but is typically 10–100 mL. The transport time to the laboratory should be minimal (<30 min) and, once it is in the laboratory, the specimen should be processed as soon as possible. If a delay cannot be avoided, the fluid should be stored in the refrigerator. Specimen Processing To process the protected brush specimen, the fluid in which the brush is suspended is agitated on a vortex mixer and the resulting suspension is used for a cytospin preparation and for culture inoculum. Using a cali- brated 0.01 mL inoculating loop, the suspension is plated onto appropriate media and carefully streaked for isolation. Colony counts of more than 1000 CFU/mL of potential pathogens (corresponding to 106 organisms/ mL of the original specimen) appear to correlate with infection (Baselski, 1994). The bronchoalveolar sample is inoculated onto agar media by using a 0.001-mL calibrated inoculating loop (as used for urine cultures, described in the following section). The presence of more than 10,000 CFU/mL of fluid correlates with disease. Staining cytocentrifuge preparations of the fluid with Gram stain is recommended; visualizing one or more bacteria without squamous epithelial cells per oil immersion field strongly suggests acute bacterial pneumonia (Kahn, 1987; Baselski, 1994). Processing bronchoalveolar lavage specimens for detection of viruses includes direct microscopic examination and conventional cell culture. Examination of cytocentrifuge preparations stained with the Papanicolaou stain allows detection of cytopathic changes, especially useful for diagnosis of CMV pneumonia (Fig. 63-4) (Woods, 1990). Cytospin preparations also may be stained with an acid-fast stain; with specific antibodies, such as those for detection of Legionella species or P. jirovecii; or with nonspecific stains (e.g., silver stain, Calcofluor white, or Giemsa) for detection of P. jirovecii or other fungi. For detection of mycobacteria, the specimen should be decontaminated and handled as described in Chapter 60. To recover fungi, the sediment of a centrifuged specimen should be inoculated on to primary fungal media.
