Diagnostic Microbiology

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Chapter 164 Diagnostic Microbiology

Anita K.M. Zaidi, Donald A. Goldmann

Laboratory diagnosis of infectious diseases is based on one or more of the following: direct examination of specimens by microscopic or antigenic techniques, isolation of microorganisms in culture, serologic testing for development of antibodies (serodiagnosis), and molecular detection of the pathogen’s genome (DNA, RNA). Clinicians must select the appropriate tests and specimens and, when possible, suggest the suspected etiologic agents to the microbiologist, because this information facilitates selection of the most cost-effective diagnostic approach. Additional roles of the microbiology laboratory include testing for antimicrobial drug susceptibility and assisting the hospital epidemiologist in detecting and clarifying the epidemiology of nosocomial infections.

Laboratory Diagnosis of Bacterial and Fungal Infections

Diagnosis of bacterial and fungal infections relies mainly on direct demonstration of microorganisms by microscopic examination or antigen detection and on growth of microorganisms on nutrient culture media. Molecular diagnostic methods for direct detection of certain pathogens are available for an increasing number of pathogens.

Microscopy

The Gram stain remains an extremely useful diagnostic technique because it is a rapid, inexpensive method for demonstrating the presence of bacteria and fungi, as well as inflammatory cells. A preliminary assessment of the etiologic agent can be made by noting the morphology (cocci vs rods) and the color (gram-positive is blue, gram-negative is red) of the microorganisms. The presence of inflammatory and epithelial cells can be used to gauge the quality of certain specimens. For example, presence of ≥10 epithelial cells per low-power field in a sputum sample strongly suggests contamination from oral secretions. In many cases the Gram stain can provide very rapid and useful results, such as in the examination of cerebrospinal fluid (CSF). The Gram stain is an insensitive technique, requiring 10⁴-10⁵ microorganisms per milliliter for detection. A trained observer may be able to reach a tentative conclusion that there are specific microorganisms in the specimen based on their morphology and staining properties (gram-positive cocci in clusters are likely to be staphylococci), but such preliminary interpretations should be made cautiously and must be confirmed by culture. Many different stains are used in clinical microbiology (Table 164-1).

Table 164-1 STAINS USED FOR MICROSCOPIC EXAMINATION

TYPE OF STAIN	CLINICAL USE
Gram stain	Stains bacteria, fungi, leukocytes, and epithelial cells
Potassium hydroxide (KOH)	A 10% solution dissolves cellular and organic debris and facilitates detection of fungal elements
Calcofluor white stain	Nonspecific fluorochrome that binds to cellulose and chitin in fungal cell walls
Calcofluor white stain	Can be combined with 10% KOH to dissolve cellular material
Ziehl-Neelsen and Kinyoun stains	Acid-fast stains, using basic carbolfuchsin, followed by acid-alcohol decolorization and methylene blue counterstaining
	Acid-fast organisms (e.g., Mycobacterium, Cryptosporidium, and Cyclospora) resist decolorization and stain pink
	A weaker decolorizing agent is used for partially acid-fast organisms (e.g., Nocardia)
Acridine orange stain	Fluorescent dye that intercalates into DNA
Acridine orange stain	At acid pH, bacteria and fungi stain orange, and background cellular material stains green
Auramine-rhodamine stain	Acid-fast stain using fluorochromes that bind to mycolic acid in mycobacterial cell walls and resist acid-alcohol decolorization
Auramine-rhodamine stain	Acid-fast organisms stain orange-yellow against a black background
India ink stain	Detects Cryptococcus neoformans, an encapsulated yeast, by excluding ink particles from the polysaccharide capsule
India ink stain	Direct testing of specimens for cryptococcal antigen is much more sensitive than India ink preparations
Methenamine silver stain	Stains fungal elements and Pneumocystis cysts in tissues
Methenamine silver stain	Primarily performed in surgical pathology laboratories
Lugol iodine stain	Added to wet preparations of fecal specimens for ova and parasites to enhance contrast of the internal structures (nuclei, glycogen vacuoles)
Wright and Giemsa stains	Primarily for detecting blood parasites (Plasmodium, Babesia, and Leishmania) and fungi in tissues (yeasts, Histoplasma)
Trichrome stain	Stains stool specimens for identification of protozoa
Direct fluorescent-antibody stain	Used for direct detection of a variety of organisms in clinical specimens by using specific fluorescein-labeled antibodies (e.g., Bordetella pertussis, Legionella, Chlamydia trachomatis, Pneumocystis jiroveci, many viruses)

Rapid Antigen Detection

Several rapid antigen detection tests for bacterial pathogens are commercially available and widely used. These include latex agglutination (LA) tests for detection of Haemophilus influenzae type b, Streptococcus pneumoniae, group B streptococci, and Neisseria meningitidis in CSF and group A streptococci in the pharynx (see later). Routinely performing LA tests on CSF is expensive and offers no advantage over an adequately performed Gram stain. Their use is best limited to patients with CSF pleocytosis who have received prior antimicrobial therapy.

A rapid and sensitive test for detecting pneumococcal antigen in the urine of patients with invasive pneumococcal disease (Binax NOW Urinary Antigen Test, Binax, Scarborough, ME) is available in the USA. A major limitation of the Binax urinary test in children is its inability to distinguish between nasopharyngeal carriage and invasive disease, because many well children who are merely colonized with pneumococci also test positive. Binax is useful in detecting pneumococcal antigen in the CSF of patients with meningitis.

Direct antigen detection tests for detecting bacterial pathogens in stools are also available. Campylobacter jejuni antigen in stool can be rapidly detected by use of a commercially available enzyme immunoassay (EIA). Similarly, Clostridium difficile toxin A and toxin B can be detected by commercially available EIA kits. The C. difficile toxins degrade rapidly at room temperature, and thus specimens must be transported promptly or refrigerated at 4°C until testing is performed for optimal test results.

Rapid antigen detection is also available for some fungal pathogens such as Cryptococcus neoformans, Candida, and Aspergillus fumigatus. Assays to detect galactomannan, a cell wall molecule in Aspergillus, are commercially available and increasingly used in the diagnosis of invasive aspergillosis in immunocompromised populations. Mannan antigen and (1,3)β-D-glucan have utility in rapid detection of invasive candidiasis.

Isolation and Identification

Most medically important bacteria can be cultured on nutrient-rich media such as blood agar and chocolate agar. Specialized agar may be used selectively to grow and differentiate among organisms of different types. For example, MacConkey agar supports growth of gram-negative rods while suppressing gram-positive organisms, and a color change in the media from clear to pink distinguishes lactose-fermenting organisms from other gram-negative rods. Broth (liquid) media are used for blood cultures and to enhance growth of small numbers of organisms in other clinical specimens. Sabouraud dextrose agar (with antibiotics to inhibit bacterial growth) is used to culture most fungi. Many pathogens, including Bartonella, Bordetella pertussis, Brucella, Francisella, Legionella, Mycoplasma, Chlamydia, and mycobacteria, and certain fungal pathogens such as Malassezia furfur require specialized growth media or incubation conditions. Consultation with the laboratory is advised when these pathogens are suspected.

After isolation in culture, microbial identity can be confirmed by a series of biochemical tests, by the ability of the organism to grow in the presence of certain substances that inhibit growth of other microorganisms (selective antibiotics, salt, bile), or by antigen detection. Molecular probes can also be used.

Blood Culture

Several different blood culture systems are available. Most use 50-100 mL bottles containing broth that enhances the growth of bacteria and fungi (mainly yeast). Bottles with smaller volumes are also available specifically for pediatric use. Media that contain resins are often used to adsorb antibiotics that may be present in a patient’s blood and to improve microbial detection. Most laboratories use automated systems that greatly reduce the time to microbial detection; >80% of all cultures containing pathogens become positive within 24 hr of incubation.

Proper skin disinfection is essential before blood is collected. Povidone-iodine may be used, but this agent must be allowed to dry completely for maximum activity. Alcohol is rapidly bactericidal and is a suitable alternative disinfectant. Iodine is effective but must be wiped off with alcohol to prevent skin reactions. The practice of obtaining blood for culture from intravascular catheters without accompanying peripheral venous blood cultures should be discouraged because it is difficult to determine the significance of coagulase-negative staphylococci and other skin flora isolated from blood obtained from “through-the-line” cultures. Differential time to positivity of 2 hr or more between paired blood cultures drawn simultaneously from a catheter and peripheral vein is a useful indicator of catheter-related bloodstream infection.

For patients with suspected bacteremia or fungemia, 2 or 3 separate blood cultures are preferred. More than 3 blood cultures rarely are indicated, even in endocarditis. Whenever possible, at least 2-3 mL of blood should be obtained for culture before antibiotics are administered. Obtaining a larger volume of blood is necessary to maximize yield from blood cultures, because children can have low-grade bloodstream infections.

For most patients, the most effective approach is to culture the entire volume of blood in a single aerobic bottle because anaerobic bacteremia is rare in children. Blood should also be cultured anaerobically for patients at increased risk for anaerobic sepsis, such as children who are immunocompromised or who have head and neck or abdominal infections. Detection of fungi can be aided by lysis-centrifugation techniques, such as the Isolator 1.5 system (Wampole, Cranbury, NJ).

Cerebrospinal Fluid Culture

CSF should be transported quickly to the laboratory and then centrifuged to concentrate organisms for microscopic examination. CSF is routinely plated on blood agar and chocolate agar, which support the growth of common pathogens causing meningitis. If tuberculosis is suspected, cultures for mycobacteria should be specifically requested. Culture of larger volumes of CSF (>5 mL) significantly improves yield of mycobacteria from CSF.

Urine Culture

Urine for culture and colony count can be obtained by collecting clean-voided midstream specimens, by catheterization, or by suprapubic aspiration. Urine samples collected by placing bags on the perineum are unacceptable for culture because samples are often contaminated, rendering the results uninterpretable. Rapid transport of urine to the laboratory is imperative because gram-negative enteric pathogens have generation times of 20-30 min, and any delay in transport or plating renders colony counts unreliable. Refrigeration may be used when delay is unavoidable. Culture systems using media-coated paddles permit prompt inoculation of the specimen, but confluent growth may be difficult to detect; moreover, accurate antibiotic susceptibility testing requires the presence of individual discrete colonies. Urine obtained by suprapubic puncture is normally sterile. Urine collected by catheterization is likely to reflect infection if there are ≥10³ organisms/mL. Clean-voided urine is considered abnormal if ≥10⁵ organisms/mL are present and possibly abnormal if 10⁴-10⁵ organisms/mL are present. However, lower counts are sometimes found in urinary tract infections in adolescent girls and young women, especially those with bacterial urethritis, or in patients with fungal infections. A Gram stain of unspun urine with ≥1 bacterium per oil immersion field correlates well with the presence of ≥10⁵ organisms/mL.

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