Jawetz Melnick & Adelberg's Medical Microbiology, 28e PDF

SECTION VII DIAGNOSTIC MEDICAL MICROBIOLOGY AND CLINICAL CORRELATION 47...

SECTION VII DIAGNOSTIC MEDICAL MICROBIOLOGY AND CLINICAL CORRELATION 47 C H A P T E R Principles of Diagnostic Medical Microbiology Diagnostic medical microbiology is concerned with the etiologic COMMUNICATION BETWEEN diagnosis of infection. Laboratory procedures used in the diagnosis of infectious disease in humans include the following: PHYSICIAN AND LABORATORY Diagnostic microbiology encompasses the detection and 1. Morphologic identification of the agent in stains of speci- characterization of thousands of agents that cause or are mens or sections of tissues (light and electron microscopy). associated with infectious diseases. The techniques used to 2. Detection of the agent in patient specimens by antigen characterize infectious agents vary greatly depending on the testing (latex agglutination, enzyme immunoassay, etc) clinical syndrome and the type of agent being considered, or nucleic acid testing (nucleic acid hybridization, poly- whether virus, bacterium, fungus, or parasite. Due to the merase chain reaction [PCR], sequencing, etc). wide variety of available tests for diagnosis of infectious dis- 3. Culture isolation and identification of the agent. Suscepti- eases and need for their interpretation, clinical information bility testing of the agent by culture or nucleic acid meth- is much more important for diagnostic microbiology than it ods, where appropriate. is for clinical chemistry or hematology. The clinician must 4. Demonstration of meaningful antibody or cell-mediated make a tentative diagnosis rather than wait until laboratory immune responses to an infectious agent. results are available. When tests are requested, the physician should inform the laboratory staff of the tentative diagnosis In the field of infectious diseases, laboratory test results (type of infection or infectious agent(s) suspected). Proper depend largely on the quality of the specimen, the timing and labeling of specimens includes such clinical data as well as the care with which it is collected and transported, and the the patient’s identifying data (at least two methods of defini- technical proficiency and experience of laboratory person- tive identification) and the requesting physician’s name and nel. Although physicians should be competent to perform a pertinent contact information. few simple, crucial microbiologic tests (perform direct wet Many pathogenic microorganisms grow slowly, and days mounts of certain specimens, make a Gram-stained smear or even weeks may elapse before they are isolated and identi- and examine it microscopically, and streak a culture plate), the fied. Treatment cannot be deferred until this process is com- technical details of the more involved procedures are usually plete. After obtaining the proper specimens and informing left to trained microbiologists. Physicians who deal with infec- the laboratory of the tentative clinical diagnosis, the clinician tious processes must know when and how to take specimens, should begin treatment with drugs aimed at the organism(s) what laboratory examinations to request, and how to interpret thought to be responsible for the patient’s illness. As the labo- the results. ratory staff begins to obtain results, they will inform health This chapter discusses diagnostic microbiology for bac- care providers, who can then reevaluate the diagnosis and terial, fungal, and viral diseases. The diagnosis of parasitic clinical course of the patient and make changes in the ther- infections is discussed in Chapter 46. apeutic program as needed. This flow of information from 757 Riedel_CH47_p757-p786.indd 757 05/04/19 5:25 PM  758   SECTION VII   Diagnostic Medical Microbiology and Clinical Correlation the laboratory consists of preliminary reports of the results 4. The specimen must be taken to the laboratory and exam- of individual steps in the isolation and identification of the ined promptly. Special transport media may be needed. causative agent. 5. Meaningful specimens to diagnose bacterial and fungal infections must be secured before antimicrobial drugs are administered. If antimicrobial drugs are given before specimens are taken for microbiologic study, drug therapy DIAGNOSIS OF BACTERIAL AND may have to be stopped and repeat specimens obtained FUNGAL INFECTIONS several days later. Specimens The type of specimen to be examined is determined Laboratory examination usually includes microscopic review by the presenting clinical picture. If symptoms or signs of fresh stained materials and inoculation of cultures with point to involvement of one organ system, specimens are conditions suitable for growth of a wide variety of microor- obtained from that source. In the absence of localizing signs ganisms, including the type of organism(s) most likely to be or symptoms, repeated blood samples for culturing are taken causative based on clinical evidence. If a microorganism is first, and specimens from other sites are then considered in isolated, species identification may then be pursued. Isolated sequence, depending in part on the likelihood of involvement microorganisms may be tested for susceptibility to antimi- of a given organ system in a given patient and in part on the crobial drugs. When significant pathogens are isolated before ease of obtaining specimens. treatment, follow-up laboratory examinations during and after treatment may be appropriate. Proper collection of the patient specimen is the single Microscopy and Stains most important step in the diagnosis of an infection, because Microscopic examination of stained or unstained specimens the results of diagnostic tests for infectious diseases depend is a relatively simple and inexpensive, but much less sensi- on the selection, timing, and method of collection of speci- tive method than culture for detection of small numbers of mens. Bacteria and fungi grow and die, are susceptible to bacteria. A specimen must contain at least 105 organisms per many chemicals, and can be found at different anatomic sites milliliter before it is likely that organisms will be seen on a and in different body fluids and tissues during the course of smear. Liquid medium containing 105 organisms per milli- infection. Because isolation of the agent is so important in the liter does not appear turbid to the eye. Specimens contain- formulation of a diagnosis, the specimen must be obtained from ing 102–103 organisms per milliliter produce growth on solid the site most likely to yield the agent at that particular stage of media, and those containing 10 or fewer bacteria per milliliter illness and must be handled in such a way as to favor the agent’s may produce growth in liquid media. survival and growth. For each type of specimen, suggestions for Gram staining is a very useful procedure in diagnos- optimal handling are given in the following paragraphs and in tic microbiology. Most specimens submitted when bac- the later section on diagnosis by anatomic site. terial infection is suspected should be smeared on glass Recovery of bacteria and fungi is most significant if the slides, Gram-stained, and examined microscopically. The agent is isolated from a site normally devoid of microorgan- materials and method for Gram staining are outlined in isms (a normally sterile area). Any type of microorganism Table 47-1. On microscopic examination, the Gram reac- cultured from blood, cerebrospinal fluid (CSF), joint fluid, tion (purple-blue indicates Gram-positive organisms; red the pleural cavity, or peritoneal cavity is a significant diagnos- indicates Gram-negative) and morphology (shape: cocci, tic finding. Conversely, many parts of the body have normal rods, fusiform, or other; see Chapter 2) of bacteria should microbiota (Chapter 10) that may be altered by endogenous or be noted. In addition, the presence or absence of inflam- exogenous influences. Recovery of potential pathogens from matory cells and the type of cell are important to note and the respiratory, gastrointestinal, or genitourinary tracts; from quantify. Likewise, the presence of material that does not wounds; or from the skin must be considered in the context of appear inflammatory, such as squamous epithelial cells the normal microbiota of each particular site. Microbiologic obtained from a respiratory sample or wound, may be use- data must be correlated with clinical information in order to ful for determining the adequacy of the sample collection. arrive at a meaningful interpretation of the results. The appearance of bacteria on Gram-stained smears does A few general rules apply to all specimens: not permit identification of species. Reports of Gram- positive cocci in chains are suggestive of, but not definitive 1. The quantity of material must be adequate. for, streptococcal species; Gram-positive cocci in clusters 2. The sample should be representative of the infectious suggest a staphylococcal species. Gram-negative rods can process (eg, sputum, not saliva; pus from the underlying be large, small, or even coccobacillary. Some nonviable lesion, not from its sinus tract; a swab from the depth of Gram-positive bacteria can stain as Gram-negative. Typi- the wound, not from its surface). cally, bacterial morphology has been defined using organ- 3. Contamination of the specimen must be avoided by using isms grown on agar. However, bacteria in body fluids or only sterile equipment and aseptic technique. tissue can have highly variable morphology. Riedel_CH47_p757-p786.indd 758 05/04/19 5:25 PM  CHAPTER 47 Principles of Diagnostic Medical Microbiology    759 TABLE 47-1 Gram and Acid-Fast Staining Methods is most useful in confirming the presence of specific organ- isms such as Bordetella pertussis or Legionella pneumophila Gram-stain in colonies isolated on culture media. The use of direct IF (1) Fix smear using heat or methanol. staining on specimens from patients is more difficult and (2) Cover with crystal violet stain (10–30 seconds). less specific and is largely being replaced by nucleic acid (3) Rinse with water. Do not blot. amplification techniques (NAATs). Stains such as calcofluor white, methenamine silver, (4) Counterstain with Gram’s iodine stain (10–30 seconds). Giemsa, and occasionally periodic acid-Schiff (PAS) and (5) Rinse with water. Do not blot. others are used for tissues and other specimens in which fungi (6) Decolorize with gentle agitation in 30% acetone–alcohol or parasites are present. Such stains are not specific for given (10–30 seconds, until stain no longer flows off slide). microorganisms, but they may define structures so that mor- (7) Rinse with water. Do not blot. phologic criteria can be used for identification. Calcofluor (8) Cover with safranin stain (10–30 seconds). white binds to cellulose and chitin in the cell walls of fungi (9) Rinse with water and air or blot dry. and fluoresces under long-wavelength ultraviolet light. It may demonstrate morphology that is diagnostic of the species (eg, Ziehl-Neelsen acid-fast stain spherules with endospores in Coccidioides immitis infection). (1) Fix smear using heat. Pneumocystis jirovecii cysts are identified morphologically in (2) Cover with carbolfuchsin, stain gently for 5 minutes over direct silver-stained specimens. PAS is used to stain tissue sections flame (or for 20 minutes over a water bath). Do not permit slides when fungal infection is suspected. After primary isolation to boil or dry out. of fungi, stains such as lactophenol cotton blue are used to (3) Rinse with deionized water. examine fungal growth and to identify organisms by their (4) Decolorize in 3.0% acid–alcohol (95% ethanol and 3.0% morphology. hydrochloric acid) until only a faint pink color remains. Specimens to be examined for fungi can be examined (5) Rinse with water. after treatment with a solution of 10% potassium hydroxide, (6) Counterstain for 1 minute with Löffler’s methylene blue stain. which breaks down the tissue surrounding the fungal myce- lia to allow a better view of the hyphal forms. Phase contrast (7) Rinse with deionized water and let dry. microscopy is sometimes useful in unstained specimens. Kinyoun carbolfuchsin acid-fast stain Dark-field microscopy can be used to detect Treponema (1) Formula: 4 g basic fuchsin, 8 g phenol, 20 mL 95% alcohol, pallidum in material from primary or secondary syphilitic 100 mL distilled water. lesions or other spirochetes such as Leptospira. (2) Stain fixed smear for 3 minutes (no heat necessary) and continue as with Ziehl-Neelsen stain. Culture Systems For diagnostic bacteriology, it is necessary to use several types Specimens submitted for examination for mycobacteria of media for routine culture, particularly when the possible should be stained for acid-fast organisms. The most sensi- organisms include aerobic, facultatively anaerobic, and obli- tive fluorescent stains for mycobacteria detection, such as gately anaerobic bacteria. The specimens and culture media auramine–rhodamine, should be used. Confirmation of a used to diagnose the more common bacterial infections are positive fluorescent stain is usually performed using one listed in Table 47-2. The standard medium for specimens is of the nonfluorescent acid-fast stains, either Ziehl-Neelsen blood agar, usually made with 5% sheep blood. Most aero- stain or Kinyoun stain (Table 47-1). These stains can be bic and facultatively anaerobic organisms will grow on blood used as alternatives to the fluorescent stains for mycobac- agar. Chocolate agar, a medium containing heated blood teria in laboratories that lack fluorescence microscopy (see with or without supplements, is a second necessary medium; Chapter 23). Immunofluorescent antibody (IF) staining some organisms that do not grow on blood agar, including is useful in the identification of many microorganisms. pathogenic Neisseria and Haemophilus, will grow on choco- Such procedures are more specific than other staining tech- late agar. A selective medium for enteric Gram-negative rods niques but also more cumbersome to perform. The fluo- (either MacConkey agar or eosin–methylene blue [EMB] rescein-labeled antibodies in common use are made from agar) is a third type of medium used routinely. These agars antisera produced by injecting animals with whole organ- contain indicators that allow differentiation of lactose- isms or complex antigen mixtures. The resultant polyclonal fermenting organisms from non-lactose-fermenting organ- antibodies may react with multiple antigens on the organ- isms. Specimens to be cultured for obligate anaerobes must ism that was injected and may also cross-react with anti- be plated on anaerobic media, such as brucella agar, a highly gens of other microorganisms or possibly with human cells supplemented medium with hemin and vitamin K or a selec- in the specimen. Quality control is important to minimize tive medium containing substances that inhibit the growth nonspecific IF staining. Use of monoclonal antibodies may of enteric Gram-negative rods and facultatively anaerobic or circumvent the problem of nonspecific staining. IF staining anaerobic Gram-positive cocci. Riedel_CH47_p757-p786.indd 759 05/04/19 5:25 PM Riedel_CH47_p757-p786.indd 760 TABLE 47-2 Common Localized Bacterial Infections 760 Disease Specimen Culture Media Common Causative Agents Usual Microscopic Findings Comments Cellulitis of skin Punch biopsy BA, CA Streptococcus pyogenes, Gram-positive cocci Biopsy at leading edge of erythema may Staphylococcus aureus yield the organism Impetigo Pus, Swab BA, CA S. pyogenes, S. aureus Gram-positive cocci Often contains skin flora Skin ulcers, Punch biopsy; BA, CA, MAC/ Mixed flora Mixed flora Often contain skin flora and deep deep tissue EMB, ANA gastrointestinal flora in below-the-waist aspirate or ulcers biopsy Meningitis CSF BA, CA Neisseria meningitidis Gram-negative intracellular Adolescents, young adults diplococci Haemophilus influenzae Small Gram-negative Adolescents, young adults coccobacilli Streptococcus pneumoniae Gram-positive cocci in pairs Adolescents, young adults Group B streptococci Gram-positive cocci in pairs Infants and chains Escherichia coli, other Gram-negative rods Infants Enterobacteriaceae species Listeria monocytogenes Gram-positive rods Immunocompromised, pregnant women, infants; β-hemolytic Brain abscess Pus BA, CA, MAC/EMB, Mixed infection; anaerobic Gram-positive cocci or Specimen must be obtained surgically and ANA Gram-positive and Gram- mixed flora transported under anaerobic conditions negative cocci and rods, aerobic Gram-positive cocci Perioral abscess Pus BA, CA, MAC/EMB, S. aureus, S. pyogenes, Mixed flora Usually mixed bacterial infection; often ANA Actinomyces contains oral flora Pharyngitis Swab BA, CA, special S. pyogenes Not recommended β-Hemolytic media for Corynebacterium diphtheriae C. diphtheriae Not recommended Clinically suspected cases; diphtheroid toxicity testing required Whooping cough NP swab, nasal Regan-Lowe agar Bordetella pertussis Not recommended Fluorescent antibody test identifies (pertussis) wash/aspirate, organisms from culture and BAL occasionally in direct smears; PCR more sensitive than culture Epiglottitis Swab BA, CA H. influenzae Usually not helpful H. influenzae is part of normal microbiota in nasopharynx 05/04/19 5:25 PM Riedel_CH47_p757-p786.indd 761 Pneumonia Sputum, BAL BA, CA, MAC/EMB S. pneumoniae Many PMNs, Gram-positive S. pneumoniae is part of normal microbiota cocci in pairs or chains. in nasopharynx. Blood cultures positive Capsule can be seen in 10–20% of patients S. aureus Gram-positive cocci in Uncommon cause of pneumonia. Usually clusters β-hemolytic, coagulase-positive Enterobacteriaceae and Gram-negative rods Hospital-associated pneumonia. Alcoholic other Gram-negative rods pneumonia Add ANA Mixed anaerobes and Mixed respiratory tract flora; Aspiration pneumonia, often associated aerobes sometimes many PMNs with pleural effusion/abscess Chest empyema Pleural fluid BA, CA. MAC/EMB, Same as pneumonia, or Mixed flora Usually pneumonia; mixed aerobic ANA mixed flora infection and anaerobic flora derived from oropharynx Liver abscess Abscess fluid BA, CA, MAC/EMB, E. coli; Bacteroides fragilis; Gram-negative rods and Commonly enteric Gram-negative aerobes ANA mixed aerobic or mixed flora and anaerobes; consider Entamoeba anaerobic flora histolytica infection Cholecystitis Bile BA, CA, MAC/EMB, Gram-negative enteric Gram-negative rods Usually Gram-negative rods from ANA aerobes, also B. fragilis gastrointestinal tract Abdominal or Abscess fluid BA, CA, MAC/EMB, Gastrointestinal flora Mixed flora Aerobic and anaerobic bowel flora; often perirectal ANA more than five species grown abscess Enteric fever, Blood, stool, BA, CA, MAC/EMB, Salmonella serovar Typhi Not recommended Multiple specimens should be cultured; typhoid urine Hektoen, enteric lactose negative, H2S positive agar Enteritis, Stool MAC/EMB, Hektoen, Salmonella species Gram-stain or methylene blue Non-lactose-fermenting colonies on enterocolitis, enteric agar, stain may show PMNs TSI slants: Nontyphoid salmonellae bacterial Campylobacter produce acid and gas in butt, alkaline diarrheas agar slant, and H2S Shigella species Gram-stain or methylene blue Non-lactose-fermenting colonies on TSI stain may show PMNs slants: Shigellae produce alkaline slant, acid butt without gas or H2S Campylobacter jejuni “Gull wing–shaped” Gram- Incubate at 42°C in Campylobacter gas; negative rods and often colonies oxidase-positive; smear shows PMNs “gull wing–shaped” rods Add TCBS agar; Vibrio cholerae Not recommended Clinically suspected cases; yellow colonies on TCBS. V. cholerae is oxidase-positive Add TCBS agar Other Vibrio species Not recommended Differentiate from V. cholerae by biochemical and culture tests Add CIN agar Yersinia enterocolitica Not recommended Enrichment at 4°C helpful; incubate cultures at 25°C (Continued) 05/04/19 5:25 PM 761 Riedel_CH47_p757-p786.indd 762 TABLE 47-2 Common Localized Bacterial Infections (Continued) 762 Disease Specimen Culture Media Common Causative Agents Usual Microscopic Findings Comments Hemorrhagic Stool MAC/EMB, E. coli O157:H7 and other Not recommended Look for sorbitol-negative colonies; type colitis and MacConkey serotypes with antisera for O antigen 157 and hemolytic sorbitol agar flagellar antigen 7; also EIAs or PCR for uremic shiga-like toxins syndrome Urinary tract Urine (clean catch BA, MAC/EMB E. coli; Enterobacteriaceae species; Gram-negative rods seen Semiquantitative culture for urine bacterial infection midstream, other Gram-negative rods on stained smear of load; E. coli Gram-negative rods bladder uncentrifuged urine indicate indole-positive; others require further catheterization more than 105 organisms/mL biochemical tests; urinalysis shows or suprapubic leukocytes and/or nitrates present aspiration) Urethritis/ Swab BA, CA, Modified Neisseria gonorrhoeae Intracellular Gram-negative Positive stained smear diagnostic in men. cervicitis Thayer-Martin agar diplococci in PMNs. Specific Nucleic acid tests more sensitive than for N. gonorrhoeae in men; culture less reliable in women Culture rarely Chlamydia trachomatis PMNs with no associated Nucleic acid tests more sensitive than performed Gram-negative diplococci culture Genital ulcers Swab, lymph BA, CA, modified Haemophilus ducreyi Mixed flora Difficult to culture, diagnosis often made node aspirate Thayer-Martin agar (chancroid) clinically Treponema pallidum Dark-field or fluorescent Culture not performed, diagnosis made (syphilis) antibody examination serologically (rapid plasma reagin [RPR], shows spirochetes but rarely venereal disease research laboratory available [VDRL] test, specific treponemal antibody tests) C. trachomatis, lymphogranuloma PMNs with no associated Diagnosis made with nucleic acid test for venereum (LGV) serovars Gram-negative diplococci C. trachomatis, LGV serovars diagnosed serologically Pelvic Cervical swab, BA, CA, modified N. gonorrhoeae PMNs with associated Nucleic acid tests more sensitive than inflammatory pelvic aspirate Thayer-Martin Gram-negative diplococci; culture disease agar mixed flora may be present C. trachomatis PMNs without associated Nucleic acid test more sensitive than Gram-negative diplococci culture Add ANA Mixed flora Mixed flora Usually mixed anaerobic and aerobic bacteria Arthritis Synovial fluid BA, CA S. aureus Gram-positive cocci in clusters Coagulase-positive; usually β-hemolytic Add modified Thayer- N. gonorrhoeae Gram-negative diplococci in Martin medium PMNs Add ANA Others Variable morphology Includes streptococci, Gram-negative rods, and anaerobes ANA, anaerobe agar (Brucella agar); BA, blood agar; BAL, bronchoalveolar lavage fluid; CA, chocolate agar; CIN, cefsulodin-irgasan-novobiocin medium; EIA, enzyme immunoassay; EMB, eosin-methylene blue agar; MAC, MacConkey agar; TCBS, thiosulfate-citrate-bile salts-sucrose agar; TSI, triple sugar iron agar. 05/04/19 5:25 PM  CHAPTER 47 Principles of Diagnostic Medical Microbiology    763 Many other specialized media are used in diagnostic bac- specific for the antigen in question, to the wells of plastic teriology; choices depend on the clinical diagnosis and the microdilution trays. The specimen containing the antigen is organism under consideration. The laboratory staff selects the incubated in the wells followed by washing of the wells. A sec- specific media on the basis of the information in the culture ond antibody for the antigen, labeled with enzyme, is used to request. Thus, freshly made Bordet-Gengou or charcoal- detect the antigen. Addition of the substrate for the enzyme containing medium is used to culture for B. pertussis in the allows detection of the bound antigen by colorimetric reac- diagnosis of whooping cough, and other special media are used tion. A significant modification of EIAs is the development to culture for Vibrio cholerae, Corynebacterium diphtheriae, of immunochromatographic membrane formats for antigen Neisseria gonorrhoeae, and Campylobacter species. For culture detection. In this format, a nitrocellulose membrane is used of mycobacteria, specialized solid and liquid media are com- to absorb the antigen from a specimen. A colored reaction monly used. These media may contain inhibitors of other bac- appears directly on the membrane with sequential addition teria. Because many mycobacteria grow slowly, the cultures of conjugate followed by substrate. In some formats, the anti- must be incubated and examined periodically for weeks (see gen is captured by bound antibody directed against the anti- Chapter 23). gen. These assays have the advantage of being rapid and also Broth cultures in highly enriched media are important frequently include a built-in internal control. EIAs are used for back-up cultures of biopsy tissues and body fluids such to detect viral, bacterial, chlamydial, protozoan, and fungal as CSF. Broth cultures may give positive results when there antigens in a variety of specimen types such as stool, CSF, is no growth on solid media because of the small number of urine, and respiratory samples. Examples of these are dis- bacteria present in the inoculum (see above). cussed in the chapters on the specific etiologic agents. Many yeasts will grow well on blood agar. Biphasic and In latex agglutination tests, an antigen-specific antibody mycelial phase fungi grow better on media designed specifi- (either polyclonal or monoclonal) is fixed to latex beads. cally for fungi. Brain–heart infusion agar, with and without When the clinical specimen is added to a suspension of the antibiotics, and inhibitory mold agar have largely replaced the latex beads, the antibodies bind to the antigens on the micro- traditional use of Sabouraud’s dextrose agar to grow fungi. organism forming a lattice structure, and agglutination of Media made with plant and vegetable materials, the natu- the beads occurs. Coagglutination is similar to latex agglu- ral habitats for many fungi, also grow many fungi that cause tination except that staphylococci rich in protein A (Cowan infections. Cultures for fungi are commonly done in paired I strain) are used instead of latex particles; coagglutination is sets, one set incubated at 25–30°C and the other at 35–37°C less useful for antigen detection compared with latex agglu- to distinguish dimorphic fungi. Table 47-3 outlines specimens tination but is helpful when applied to identification of bac- and other tests to be used for the diagnosis of fungal infections. teria in cultures such as Streptococcus pneumoniae, Neisseria In addition to the above standard and selective media, meningitidis, N. gonorrhoeae, and β-hemolytic streptococci. agars that incorporate antibiotics and chromogenic enzyme Latex agglutination tests are primarily directed at the substrates that impart color to specific organisms of inter- detection of carbohydrate antigens of encapsulated microor- est, such as methicillin-resistant Staphylococcus aureus and ganisms. Antigen detection is used most often in the diagnosis various Candida species, among many others, are available. of group A streptococcal pharyngitis. Detection of cryptococ- These media, while more expensive, do enhance sensitivity by cal antigen is useful in the diagnosis of cryptococcal meningitis inhibiting background microbiota and allowing the pathogen in patients with AIDS or other immunosuppressive diseases. of interest to be more easily recognized. Typically, these chro- The sensitivity of latex agglutination tests in the diagnosis mogenic agars are used for specimens such as surveillance of bacterial meningitis is not better than that of Gram-stain, cultures and cultures of urine. which is approximately 100,000 bacteria per milliliter. For that reason, the latex agglutination test is not recommended for direct CSF specimen testing. Antigen Detection Immunologic systems designed to detect antigens of micro- organisms can be used in the diagnosis of specific infections. Serological Testing Immunofluorescent tests (direct and indirect fluorescent Detection of specific antibodies to infectious agents can be antibody tests) are one form of antigen detection and are useful for diagnosis of acute or chronic infections, and for discussed in separate sections in this chapter and in the investigating the epidemiology of infectious disease. During chapters on the specific microorganisms. the course of illness, IgM antibody is first produced, followed Enzyme immunoassays (EIAs), including enzyme- by appearance of IgG antibody. Caution must be used when linked immunosorbent assays (ELISA), and agglutination interpreting positive IgM results, as these assays demonstrate tests are used to detect antigens of infectious agents present in cross-reactivity and can be falsely positive. Serology is most clinical specimens. The principles of these tests are reviewed useful when acute and convalescent sera are tested to show briefly here. increases in antibody titers over time. There are many variations of EIAs to detect antigens. There are a variety of serological assays available, includ- One commonly used format is to bind a capture antibody, ing direct immunofluorescence, agglutination, complement Riedel_CH47_p757-p786.indd 763 05/04/19 5:25 PM Riedel_CH47_p757-p786.indd 764 TABLE 47-3 Common Fungal Infections and Nocardiosis: Agents, Specimens, and Diagnostic Tests 764 Specimen Serologic and Other Tests Comments Invasive (deep-seated) mycoses Aspergillosis: Aspergillus fumigatus, other Aspergillus species Pulmonary Sputum, BAL Culture, serum/BAL Must distinguish colonization from infection galactomannan assays Disseminated Biopsy specimen, blood As above Aspergillus is difficult to grow from blood and body fluids of patients with disseminated infection. Blastomycosis: Blastomyces dermatitidis Pulmonary Sputum, BAL Culture, serology Serology useful to determine exposure; definitive diagnosis requires culture; yeast show broad-based budding. Oral and cutaneous ulcers Biopsy or swab specimen Culture, serology Bone Bone biopsy Culture, serology Coccidioidomycosis: Coccidioides immitis Pulmonary Sputum, BAL Culture, serology Serology often more sensitive than culture; positive immunodiffusion can be followed by complementation fixation titers; C. immitis can grow on routine bacterial cultures and pose laboratory exposure risk. Disseminated Biopsy specimen, CSF As above Histoplasmosis: Histoplasma capsulatum Pulmonary Sputum, BAL Culture, serology, urine antigen Serology useful to determine exposure; definitive diagnosis test requires culture. Disseminated Bone marrow, biopsy specimen As above Pathology shows small intracellular yeast forms distinguished from Leishmania by the absence of kinetoplast. Nocardiosis: Nocardia asteroides complex and other Nocardia species Pulmonary Sputum, BAL Culture, modified acid-fast stain Nocardiae are bacteria that clinically behave like fungi; weakly acid-fast, branching, filamentous Gram-positive rods. Subcutaneous Aspirate or biopsy of abscess Brain Material from brain abscess Paracoccidioidomycosis (South American blastomycosis): Paracoccidioides brasiliensis Biopsy specimen Culture, serology Serology useful to determine exposure; definitive diagnosis requires culture. Sporotrichosis: Sporothrix schenckii Skin and subcutaneous nodules Biopsy specimen Culture, serology Soil and gardening exposure Disseminated Biopsy specimen 05/04/19 5:25 PM Riedel_CH47_p757-p786.indd 765 Zygomycosis: Rhizopus species, Mucor species, others Rhinocerebral Nasal-orbital tissue Culture Nonseptate hyphae seen in microscopic sections Cutaneous; pulmonary and disseminated Sputum, BAL, biopsy specimens Culture Yeast infections Candidiasis: Candida albicans and other Candida speciesa Mucous membrane Oral swab, vaginal swab, biopsy Culture, yeast wet mount Vaginal candidiasis diagnosed clinically and by Gram-stain specimen (Nugent criteria) Skin Swab, biopsy specimen Culture Systemic Blood, biopsy specimen, urine Culture Candida and other yeast species grow well in routine bacterial cultures. Cryptococcosis: Cryptococcus neoformans Pulmonary Sputum, BAL Culture, cryptococcal antigen Most common in immunocompromised patients Meningitis CSF Culture, cryptococcal antigen Disseminated Bone marrow, bone, blood, other Culture, cryptococcal antigen Primary skin infections Dermatophytosis: Microsporum species, Epidermophyton species, Trichophyton species Hair, skin, nails from infected sites Culture Requires specialized dermatophyte agars a Candida tropicalis, Candida parapsilosis, Candida glabrata, and other Candida species. BAL, bronchoalveolar fluid; CF, complement fixation; CSF, cerebrospinal fluid; EIA, enzyme immunoassays. 05/04/19 5:25 PM 765  766   SECTION VII   Diagnostic Medical Microbiology and Clinical Correlation fixation (CF), EIA, and ELISA formats. There are also non- (normally negatively charged) is replaced by a polyamide of specific immunoassays available, such as the heterophile test repetitive units (neutral charge). Individual nucleotide bases for EBV mononucleosis and rapid plasma reagin for syphilis. can be attached to the now neutral backbone, which allows Several of these tests can measure antibody titer by perform- for faster and more specific hybridization to complementary ing dilutions of patient serum to determine the lowest titer at nucleic acids. Because the probes are synthetic, they are not which reactivity is seen. subject to degradation by nucleases and other enzymes. These probes can be used for detection of S. aureus, enterococci, cer- tain Candida spp., and some Gram-negative bacilli from posi- Western Blot Immunoassays tive blood culture bottles. The probe hybridization is detected These assays are usually performed to detect antibodies against by fluorescence and is called peptide nucleic acid–fluorescence specific antigens of a particular organism. This method is in situ hybridization (PNA-FISH). based on the electrophoretic separation of major proteins of the organism in question in a two-dimensional agarose gel. B. Microbial Identification Using Ribosomal Gene Organisms are mechanically or chemically disrupted, and resultant solubilized antigen of the organism is placed in a Probe Hybridization polyacrylamide gel. An electric current is applied, and major The ribosomal RNA (rRNA) genes of each species has stable proteins are separated out on the basis of size (smaller pro- (conserved) portions of the sequence. Most assays target the teins travel faster). The protein bands are transferred to strips bacterial 16S rRNA gene or the internal transcribed spacer of nitrocellulose paper. Following incubation of the strips with regions of the fungal rRNA genes. Many copies are present in a patient’s specimen containing antibody (usually serum), the each organism. Labeled probes specific for the rRNA gene of antibodies bind to the proteins on the strip and are detected a species are added, and the amount of label on the double- enzymatically in a fashion similar to the EIA methods stranded hybrid is measured. This technique is widely used described earlier. Western blot tests are used as specific tests for the rapid identification of many organisms. Examples for antibodies in HIV infection and Lyme disease. include the most common and important Mycobacterium species, C. immitis, Histoplasma capsulatum, and others. Molecular diagnostic assays that use amplification Molecular Diagnostics of nucleic acid have become widely used and are evolving A. Nucleic Acid Hybridization Probes rapidly. They have been used on a variety of sample types, including direct patient specimens, positive cultures, and iso- The principle behind hybridization probe molecular assays is lated organisms. These amplification systems fall into several the hybridization of a characterized nucleic acid probe to a basic categories as outlined below. specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. For example, single-stranded probe DNA (or RNA) is used to detect complementary RNA C. Target Amplification Systems or denatured DNA in a test specimen. The nucleic acid In these assays, the target DNA or RNA is amplified many probe typically is labeled with enzymes, antigenic substrates, times. The polymerase chain reaction (PCR) is used to chemiluminescent molecules, or radioisotopes to facilitate amplify extremely small amounts of specific DNA present in detection of the hybridization product. By carefully selecting a clinical specimen, making it possible to detect what were the probe or making a specific oligonucleotide and performing initially minute amounts of the DNA. PCR uses a thermo- the hybridization under conditions of high stringency, detec- stable DNA polymerase to produce a twofold amplification tion of the nucleic acid in the test specimen can be extremely of target DNA with each temperature cycle. Conventional specific. Such assays are currently used primarily for rapid PCR, also referred to as end detection PCR, utilizes three confirmation of a pathogen once growth is detected (eg, the sequential reactions—denaturation, annealing, and primer identification of Mycobacterium tuberculosis in culture using extension—as follows. The DNA extracted from the clinical a DNA probe). In situ hybridization involves the use of labeled specimen along with sequence-specific oligonucleotide prim- DNA probes or labeled RNA probes to detect complementary ers, nucleotides, thermostable DNA polymerase, and buffer nucleic acids in formalin-fixed paraffin-embedded tissues, are heated to 90–95°C to denature (separate) the two strands frozen tissues, or cytologic preparations mounted on slides. of the target DNA. The temperature in the reaction is low- Technically, this can be difficult and is usually performed in ered, usually to 45–60°C depending on the primers, to allow histology laboratories and not clinical microbiology laborato- annealing of the primers to the target DNA. Each primer ries. However, this technique has increased the knowledge of is then extended by the thermostable DNA polymerase by the biology of many infectious diseases, especially the hepa- adding nucleotides complementary to the target DNA yield- titides and oncogenic viruses, and is still useful in infectious ing the twofold amplification. The cycle is then repeated diseases diagnosis. A novel technique that is somewhat of 30–40 times to yield amplification of the target DNA segment a modification of in situ hybridization makes use of peptide by more than 1010-fold. The amplified segment often can be nucleic acid probes. Peptide nucleic acid probes are synthesized seen in an electrophoretic gel or detected by Southern blot pieces of DNA in which the sugar phosphate backbone of DNA analysis using labeled DNA probes specific for the segment Riedel_CH47_p757-p786.indd 766 05/04/19 5:25 PM  CHAPTER 47 Principles of Diagnostic Medical Microbiology    767 or by a variety of proprietary commercial techniques. More from the second primer, producing a double-stranded DNA recently, real-time PCR protocols have replaced these end copy, with intact RNA polymerase. The RNA polymerase then detection methods (see below). produces many copies of the single-stranded RNA. Detection PCR can also be performed on RNA targets, which is of C. trachomatis, N. gonorrhoeae, and M. tuberculosis and called reverse transcriptase PCR. The enzyme reverse tran- quantitation of HIV-1 viral loads are examples of the use of scriptase is used to transcribe the RNA into complementary these types of assays. DNA for subsequent PCR amplification. Strand displacement assays (SDA) are isothermal PCR assays are available commercially for identifica- amplification assays that employ use of restriction endonu- tion of a broad range of bacterial and viral pathogens, such clease and DNA polymerase. The restriction endonuclease as Chlamydia trachomatis, N. gonorrhoeae, M. tuberculosis, “nicks” the DNA at specific sites allowing DNA polymerase cytomegalovirus (CMV), HIV-1, hepatitis C virus, and many to initiate replication at the nicks on the target molecule and others. There are many other laboratory-developed PCR simultaneously displacing the nicked strand. Displaced single assays that have been implemented by individual laboratories strands then serve as templates for additional amplification. to diagnose infections. Such assays are the tests of choice to Loop-mediated isothermal amplification (LAMP) gets diagnose many infections—especially when traditional cul- its name from the fact that the final amplification product ture and antigen detection techniques do not work well. Exam- consists of a structure that contains multiple loops (repeats) ples include testing of CSF for herpes simplex virus (HSV) to of the target sequence. The reaction is isothermal and con- diagnose herpes encephalitis and testing of nasopharyngeal sists of autocycling strand displacement DNA synthesis using samples to diagnose B. pertussis infection (whooping cough). Bst DNA polymerase and four to six primers. Amplification A major consideration for laboratories that perform PCR products can be detected in real time by precipitating assays is to prevent contamination of reagents or specimens DNA by adding magnesium pyrophosphate to the reaction with target DNA from the environment, which can obscure creating turbidity that can be read visually or by using a spec- the distinction between truly positive results and falsely posi- trophotometer. This method is very sensitive, detecting as tive ones because of the contamination. few as 10 target copies per reaction. Commercial assays using LAMP technology for the detection of Clostridium difficile in D. Signal Amplification Techniques stool samples and for other pathogens in a variety of speci- men types are available. These assays strengthen the signal by amplifying the label (eg, fluorochromes, enzymes) that is attached to the target F. Real-Time PCR nucleic acid. The branched DNA (bDNA) system has a series Technologic advances, which have lead to “real-time ampli- of primary probes and a branched secondary probe labeled fication,” have streamlined nucleic acid amplification plat- with enzyme. Multiple oligonucleotide probes specific for forms, improved the sensitivity of amplification tests, and the target RNA (or DNA) are fixed to a solid surface such drastically reduced the potential for contamination. Dramatic as a microdilution tray. These are the capture probes. The improvements in the chemistry of nucleic acid amplification prepared specimen is added, and the RNA molecules are reactions have resulted in homogeneous reaction mixtures attached to the capture probes on the microdilution tray. in which fluorogenic compounds are present in the same Additional target probes bind to the target but not to the tray. reaction tube in which the amplification occurs. A variety The enzyme-labeled bDNA amplifier probes are added and of fluorogenic molecules are used. These include nonspecific attach to the target probes. A chemiluminescent substrate dyes such as SYBR green, which binds to the minor groove is added, and light emitted is measured to quantitate the of double-stranded DNA, and amplicon-specific detection amount of target RNA present. Examples of the use of this methods using fluorescently labeled oligonucleotide probes, type of assay include the quantitative measurement of HIV-1, which fall into three categories: TaqMan or hydrolysis probes, hepatitis C virus, and hepatitis B virus. fluorescence energy transfer (FRET) probes, and molecular beacons. The signal from these probes is proportional to the E. Amplification Methods: Non-PCR-Based amount of product DNA present in the reaction and is plotted The transcription-mediated amplification (TMA) and the against the PCR cycle. Use of a threshold fluorescence value nucleic acid sequence-based amplification (NASBA) sys- allows determination of positive and negative reactions. The tems amplify large quantities of RNA in isothermal assays signal is measured through the closed reaction tube using that coordinately use the enzymes reverse transcriptase, fluorescent detectors; hence, the assay is performed in “real RNase H, and RNA polymerase. An oligonucleotide primer time.” Since the reaction tube does not need to be opened to containing the RNA polymerase promoter is allowed to bind analyze the PCR products on a gel, there is much less risk to the RNA target. The reverse transcriptase makes a single- of amplicon carryover to the next reaction. When used with stranded cDNA copy of the RNA. The RNase H destroys the a standard curve, real-time PCR assays can be quantita- RNA of the RNA–cDNA hybrid, and a second primer anneals tive, allowing for determination of organism concentration. to the segment of cDNA. The DNA-dependent DNA poly- These assays are commonly used for viral load quantifica- merase activity of reverse transcriptase extends the DNA tion of HIV, hepatitis C virus, hepatitis B virus, and CMV. Riedel_CH47_p757-p786.indd 767 05/04/19 5:25 PM  768   SECTION VII   Diagnostic Medical Microbiology and Clinical Correlation The reader is referred to the Persing et al reference for more used to assemble entire organism genomes, define the micro- detailed information about real-time PCR and other molecu- biome, detect infectious agents, or look for low-level sequence lar methods. variants, known as quasispecies. Database comparison can be used to classify the organism subtype or determine the pres- G. PCR-Sequencing ence of markers of drug resistance or virulence. The product of a PCR reaction can be sequenced and com- pared to a database for identification of organisms or resis- Mass Spectrometry tance mutations. PCR primers are designed to hybridize to conserved genomic regions, with the sequence of inter- Mass spectrometry (MS), a technology used to analyze pro- est amplified between the primers. A variety of sequencing teins or DNA, has revolutionized the approach to microbial methods can be used, the discussion of which is beyond the identification in clinical laboratories. MS employs methods scope needed here. such as ionization radiation to disrupt material, forming For bacterial identification, sequencing of the 16S rRNA charged particles that are identified in various ways on the gene is commonly used. This gene has highly conserved basis of mass or mass-to-charge ratio. Applications in micro- regions interspersed with variable sequences, making it ideal biology have been made possible by advances in technology, for amplifying and differentiating many bacterial species. such as matrix-assisted laser desorption ionization time-of- Other conserved gene targets are also used for bacterial iden- flight mass spectroscopy (MALDI-TOF MS). Several differ- tification, including rpoB, sodA, and hsp65. Similarly, fun- ent methods are briefly described below. gal identification can be performed using PCR-sequencing MassTag PCR incorporates a tag of known mass (a library of 28S rRNA gene and ribosomal RNA gene internal tran- of 64 mass tags is commercially available) into the PCR prod- scribed spacer elements. uct. Most frequently used in multiplex PCR reactions, the PCR-sequencing is also used for strain typing and detec- tags are released by ultraviolet (UV) irradiation and analyzed tion of specific resistance mutations in viruses (see Diagnosis by MS. The identity of the desired target or targets is deter- of Viral Infections section below). Its use is expanding to gene mined by the size of the tag(s). characterization in other organisms, such as the detection of PCR electrospray ionization mass spectrometry (PCR- certain mutations causing rifampin or isoniazid resistance in ESI-MS) uses a unique principle. Briefly, for particular M. tuberculosis. microbes, a set of PCR primers is designed that amplifies key regions of the microbial genome. Multiple PCR reac- tions are conducted in a microtiter plate for analysis of each H. Microarrays sample, and some of the wells contain more than one primer Nucleic acid microarrays involve the use of multiple oligonu- pair. Following PCR, the microtiter plate is placed on a fully cleotide probes to detect the complementary target sequence automated instrument and ESI-MS analysis is performed. in amplified DNA or RNA. The arrays can have from tens The mass spectrometer is an analytical tool that effectively to hundreds of thousands of probes (high-density microar- weighs the amplicons, or mixture of amplicons, with suffi- rays) and yield substantial information about the genetic cient mass accuracy that the composition of A, G, C, and T makeup of specific organisms. Patient samples or clinical can be deduced for each amplicon in the PCR reaction. The isolates are subject to DNA amplification labeling followed determined composition is then interrogated using a propri- by hybridization, washing, and detection of labeled DNA etary software database. bound to specific probes. Microarrays can be used to detect The above two platforms allow for direct detection of microorganisms directly from patient samples or positive the nucleic acid of microbes directly from clinical samples blood cultures through the use of conserved targets such as without a culture step because they use a PCR amplification 16S ribosomal DNA probes. They can also provide genetic reaction. Another application is using MALDI-TOF to iden- profiling of isolated organisms, yielding information about tify bacteria and yeast isolates recovered in clinical cultures. the genotype, virulence factors, or resistance markers present These platforms target the highly abundant ribosomal pro- in the organism. teins of bacteria and yeast. The assays involve making a thin smear from a colony or broth culture onto a metallic slide of I. High-Throughput Sequencing the organism to be identified and applying an acid matrix to High-throughput sequencing (also known as next-generation it. The slide is placed into the instrument where the organ- or “deep” sequencing) involves the simultaneous sequencing ism mixture is hit by laser pulses. Charged protein fragments of a large number of DNA molecules (known as a library). are produced and accelerated through an electrostatic field The source of the library can be an organism isolate or direct in a vacuum tube until they contact the mass spectrometer’s patient sample. Several different instrument platforms are detector. Molecules of different masses and charges “fly” available, and can generate thousands to millions of sequence at different speeds (“time of flight”). A spectral signature, reads per sample. Bioinformatic algorithms are then used generally in the range of 1000–20,000 mass-to-charge ratio to classify, assemble, and compare the sequence to known (m/z), is generated. This spectral signal is compared to others organism databases. High-throughput sequencing can be in the proprietary databases of each instrument for genus or Riedel_CH47_p757-p786.indd 768 05/04/19 5:25 PM  CHAPTER 47 Principles of Diagnostic Medical Microbiology    769 species assignment of the organism. The reader is referred to a tentative etiologic diagnosis on clinical grounds. On the the Patel reference for additional details. basis of this “best guess,” a drug that is likely to be effective against the suspect agent(s) can be selected (see Chapter 28). Before this drug is administered, specimens are obtained THE IMPORTANCE OF NORMAL for laboratory detection of the causative agent. The results BACTERIAL AND FUNGAL MICROBIOTA of these examinations may allow for narrowing of antibiot- ics to targeted therapy (as opposed to broad Gram-positive Organisms, such as M. tuberculosis, Salmonella serovar Typhi, and Gram-negative coverage for sepsis). The identification of and Brucella species, are considered pathogens whenever they certain microorganisms that are uniformly drug-susceptible are found in patient specimens. However, many infections are eliminates the necessity for further testing and permits the caused by organisms that are permanent or transient members selection of optimally effective drugs based on the organ- of the normal microbiota. For example, E. coli is part of the nor- ism’s known susceptibility profile. When the organism mal gastrointestinal microbiota, but is also the most common resistance profile is varied, tests for drug susceptibility of cause of urinary tract infections. Similarly, the vast majority of isolated microorganisms will guide optimal drug choice (see mixed bacterial infections with anaerobes are caused by organ- Chapter 28). Disk diffusion susceptibility tests measure the isms that are members of the normal microbiota. ability of bacteria to grow on the surface of an agar plate in The relative number of specific organisms found in a cul- the presence of paper disks containing antibiotic drug. The ture is important when members of the normal microbiota drug diffuses out into the surrounding agar, inhibiting bac- are the cause of infection. When numerous Gram-negative terial growth in a circular area surrounding the disk. The rods of species such as Klebsiella pneumoniae are found mixed diameter of this zone of growth inhibition is measured, and with a few normal nasopharyngeal bacteria in a sputum cul- correlates with the susceptibility of the isolate being tested. ture, the Gram-negative rods are strongly suspect as the cause The choice of drugs to be included in a routine susceptibility of pneumonia because large numbers of Gram-negative rods test battery should be based on the susceptibility patterns of are not normally found in sputum or in the nasopharyngeal isolates in the laboratory, the type of infection (community- microbiota; the organisms should be identified and reported. acquired or nosocomial), the source of the infection, and In contrast, abdominal abscesses commonly contain a normal cost-effectiveness analysis for the patient population. The distribution of aerobic, facultatively anaerobic, and obligately Clinical and Laboratory Standards Institute (CLSI) (Wayne, anaerobic organisms, representative of the gastrointestinal PA) provides recommendations for which agents to test based microbiota. In such cases, identification of all species present on the organism recovered and the specimen type, and inter- is not warranted; instead, it is appropriate to report “normal pretive criteria (susceptible, intermediate, or resistant) based gastrointestinal microbiota.” on the measured zone size. Yeasts in small numbers are commonly part of the nor- The sizes of zones of growth inhibition vary with the mal microbial microbiota. However, other fungi are not pharmacologic characteristics of different drugs. Thus, the normally present and therefore should be identified and zone size of one drug cannot be compared to the zone size reported. Viruses usually are not part of the normal micro- of another drug acting on the same organism. However, for biota as detected in diagnostic microbiology laboratories, but any one drug the zone size can be compared to a standard, can be found in otherwise healthy individuals, presumably provided that media, inoculum size, and other conditions are as asymptomatic infections. Latent viruses, such as herpes carefully controlled. This makes it possible to define for each simplex or CMV, or live vaccine viruses, such as poliovirus, drug a diameter of inhibition zone that distinguishes suscep- can be detected in asymptomatic cases. In some parts of the tible from intermediate or resistant strains. world, stool specimens commonly yield evidence of parasitic The disk test measures the ability of drugs to inhibit the infection without symptoms present. Therefore, the clinical growth of bacteria in vitro. The results correlate reasonably presentation of infectious disease illness along with the rela- well with therapeutic response in those disease processes in tive number of potentially pathogenic organisms is impor- vivo when body defenses can eliminate infectious microor- tant in establishing the correct diagnosis. ganisms, but may be less well correlated with response in Members of the normal microbiota that are most com- immunocompromised patients. The selection of appropri- monly present in patient specimens and that may be reported ate antibiotic therapy depends on clinical as well as bacterial as “normal microbiota” are discussed in Chapter 10. factors, such as use of bactericidal rather than bacteriostatic drugs for endocarditis, or drugs that will penetrate the blood–brain barrier for central nervous system infections LABORATORY AIDS IN THE SELECTION (see Chapter 28). Minimum inhibitory concentration (MIC) OF ANTIMICROBIAL THERAPY tests measure the ability of organism to grow in broth culture in the presence of various dilutions of antibiotics. It measures The antimicrobial drug used initially in the treatment of an more exactly the concentration of an antibiotic necessary to infection is chosen on the basis of clinical impression after the inhibit growth of a standardized inoculum under defined clinician is convinced that an infection exists and has made conditions. A semiautomated microdilution method is used Riedel_CH47_p757-p786.indd 769 05/04/19 5:25 PM  770   SECTION VII   Diagnostic Medical Microbiology and Clinical Correlation in which defined amounts of drug are dissolved in a mea- The pus in closed, undrained soft tissue abscesses sured small volume of broth and inoculated with a standard- frequently contains only one organism as the infecting ized number of microorganisms. The end point, or MIC, is agent; most commonly staphylococci, streptococci, or enteric considered the last broth cup (lowest concentration of drug) Gram-negative rods. The same is true in acute osteomy- remaining clear, that is, free from microbial growth. The MIC elitis, where the organisms can often be cultured from provides a better estimate of the probable amount of drug blood before the infection has become chronic. Multiple necessary to inhibit growth in vivo and thus helps in gaug- microorganisms are frequently encountered in abdominal ing the dosage regimen necessary for the patient. Guidelines abscesses and abscesses contiguous with mucosal surfaces available from CLSI provide interpretive criteria, defining as well as in open wounds. When deep suppurating lesions, strains as resistant, intermediate, or susceptible to a certain such as chronic osteomyelitis, drain onto exterior surfaces drug based on the MIC. through a sinus or fistula, the microbiota of the surface The MIC only shows that bacterial growth is inhibited through which the lesion drains must not be mistaken for at that drug concentration; there may still be viable bacte- that of the deep lesion. Instead, specimens should be aspi- ria that can recover when the drug is removed. Bactericidal rated from the primary infection through uninfected tissue. effects can be estimated by subculturing the clear broth from Bacteriologic examination of pus from closed or deep MIC testing onto antibiotic-free solid media. The result, for lesions must include culture by anaerobic methods. Anaer- example, a reduction of colony-forming units by 99.9% below obic bacteria (Bacteroides, Fusobacterium, etc) sometimes that of the control, is called the minimal bactericidal con- play an essential causative role, and mixtures of aerobes and centration (MBC). anaerobes are often present. Because empiric therapy must often be given before the The methods used for cultures must be suitable for the results of antimicrobial susceptibility tests are available, it is semiquantitative recovery of common bacteria and also for recommended by CLSI that laboratories publish an antibio- recovery of specialized microorganisms, including myco- gram annually that contains the results of susceptibility test- bacteria and fungi. Eroded skin and mucous membranes are ing in aggregate for particular organism–drug combinations. frequently the sites of yeast or fungus infections. Candida, For example, it may be important to know the most active Aspergillus, and other yeasts or fungi can be seen microscopi- β-lactam antimicrobial agent targeted against Pseudomonas cally in smears or scrapings from suspicious areas and can be aeruginosa among intensive care unit patients in a particu- grown in cultures. Treatment of a specimen with KOH and lar hospital. This allows the best therapy to be chosen based calcofluor white greatly enhances the observation of yeasts on clinical suspicion of the infecting organism and known and molds in the specimen. locally circulati