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.

Genital Culture

Specimens from the genital tract include urethral, cervical, and anorectal swabs. Neisseria gonorrhoeae organisms are fragile, and rapid inoculation at the bedside onto Thayer-Martin medium (warmed to room temperature) or one of its modifications is crucial. Ordinarily, specimens for N. gonorrhoeae should be obtained from genital, anorectal, and pharyngeal sources to achieve maximum yield. Specimens for Chlamydia trachomatis culture are obtained by cotton-tipped, aluminum-shafted urethral swabs. Endocervical specimens, using swabs with aluminum or plastic shafts, should be collected by rubbing the swab vigorously against the endocervical wall to obtain as much cellular material as possible. C. trachomatis is cultured by inoculation into cell culture systems, followed by immunofluorescent staining with monoclonal antibody against the organism. Nonculture methods such as enzyme immunoassay (EIA) tests, direct immunofluorescent staining by monoclonal antibodies, and DNA amplification methods are widely used and are more cost-effective than culture.

Throat and Respiratory Cultures

Obtaining a throat swab for culture is the most reliable method for diagnosing group A streptococcal pharyngitis and tonsillitis. Vigorous swabbing of the tonsillar area and posterior pharynx is necessary for maximum detection. Even then, a single swab detects only approximately 90% of infections. The pharynx contains many normal flora; thus, most laboratories screen cultures only for the presence of group A β-hemolytic streptococci. Some laboratories do not use selective procedures, however, and may report the presence of meningococci, usually nontypable, nonpathogenic strains, but occasionally typable meningococci and other potential pathogens. Most patients harboring such bacteria are carriers, and the culture report serves only to create needless alarm. The laboratory should be alerted if diphtheria, pertussis, gonococcal pharyngitis, or infection with Arcanobacterium haemolyticum is clinically suspected. Cultures for B. pertussis are obtained by aspiration or a Dacron or flexible aluminum wire calcium alginate swab (Calgiswab) of the nasopharynx and inoculated onto special charcoal-blood (Regan-Lowe) or Bordet-Gengou media.

The cause of lower respiratory tract disease in children is not easy to confirm microbiologically because of difficulty in obtaining adequate sputum specimens and lack of correlation between upper respiratory tract flora and organisms causing lower respiratory tract disease. Gram-stained smears of specimens with large numbers of epithelial cells or with few neutrophils are unsuitable for culture. Patients with cystic fibrosis can usually provide adequate expectorated sputum, and special media should be used to detect important cystic fibrosis pathogens, such as Burkholderia cepacia.

Endotracheal aspirates from intubated patients may be useful if the Gram stain shows abundant neutrophils and bacteria, although pathogens recovered from such specimens might still reflect only contamination from the endotracheal tube or upper airway. Quantitative cultures of bronchoalveolar lavage fluid or bronchial brush specimens may be valuable for distinguishing upper respiratory tract contamination from lower tract disease in special circumstances.

The diagnosis of pulmonary tuberculosis in young children is best made by culture of early-morning gastric aspirates, obtained on 3 successive days. In young children, gastric cultures have the highest yield on the 1st day of collection. Acid-fast stains of gastric aspirates from children are rarely positive. Sputum induction for obtaining specimens for mycobacterial culture has also proved useful in young children but requires skilled personnel and sophisticated containment facilities to prevent exposure of health care workers. Cultures for Mycobacterium tuberculosis should be processed only in laboratories equipped with appropriate biologic safety cabinets and containment facilities.

Stool Culture

Most laboratories in North America routinely culture stool for the presence of Salmonella, Shigella, and Campylobacter by inoculation onto selective agar (to decrease the growth of normal fecal flora) and differential agar (to help distinguish pathogenic enteric flora from normal enteric flora). Freshly passed stool is preferred, but rectal swabs may be acceptable. Cultures for Escherichia coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, Aeromonas, and Plesiomonas should be specifically requested when these organisms are suspected. Isolation of enterotoxigenic E. coli, the most common cause of traveler’s diarrhea, and other pathogenic types of E. coli except for E. coli O157:H7 is not routinely attempted. Bacterial stool cultures in the USA rarely yield useful results in patients who have been hospitalized for more than 3 days. Cultures for C. difficile, the most common bacterial cause of nosocomial diarrhea, have been replaced by tests that detect toxin production, such as cell culture assays for cytotoxicity, and EIA.

Culture of Other Fluids and Tissues

Abscesses, wounds, pleural fluid, peritoneal fluid, joint fluid, and other purulent fluids are cultured onto routine solid agar and broth media. Whenever possible, fluid rather than swabs from infected sites should be sent to the laboratory, because culture of a larger volume of fluid can detect organisms present in low concentration. Anaerobic organisms are involved in many abdominal and wound abscesses. These specimens should be collected and transported rapidly under anaerobic conditions, preferably in anaerobic transport tubes.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility tests are generally performed on all organisms of clinical significance except for a few that have predictable antimicrobial susceptibility patterns (group A streptococci remain universally susceptible to penicillin). The most common technique is the agar disk diffusion method (Bauer-Kirby method), in which a standardized inoculum of the organism is seeded onto an agar plate. Antibiotic-impregnated filter paper disks are then placed on the agar surface. After 18-24 hr of incubation, the zone of inhibition of bacterial growth around each disk is measured and compared with nationally determined standards for susceptibility or resistance.

The other widely used technique for susceptibility testing is dilution testing. A standard concentration of a microorganism is inoculated into serially diluted concentrations of antibiotic, and the minimum inhibitory concentration (MIC) in µg/mL, the lowest concentration of antibiotic required to inhibit growth of the microorganism, is determined. Dilution testing also permits determination of the minimum bactericidal concentration (MBC), the lowest concentration of antibiotic required to kill the organism. The MBC is sometimes determined to exclude the possibility of bacterial tolerance (MBC >4 times the MIC). Automated methods that use microtiter wells with premade dilutions of antibiotics are now used commonly. However, MICs from automated systems should be interpreted with caution for certain combinations of pathogen and antibiotic (e.g., pneumococci resistant to penicillin, enterococci with low-level resistance to vancomycin). Screening agar plate tests, such as oxacillin disk susceptibility to detect penicillin-resistant pneumococci, followed by confirmatory tests, are recommended. The E-test is used to measure the MIC of individual antibiotics on an agar plate. It uses a paper strip impregnated with a known continuous concentration gradient of antibiotic that diffuses across the agar surface, inhibiting microbial growth in an elliptic zone. The MIC is read off the printed strip at the point at which the zone intersects the strip. Major advantages of the E-test are reliable interpretation, reproducibility, and applicability to organisms that require special media or growth conditions, including anaerobic bacteria.

Antimicrobial susceptibility patterns are rapidly changing as microbes evolve new resistance mechanisms. Recommendations for performance standards for antimicrobial susceptibility tests and their interpretation are regularly updated by the Clinical and Laboratory Standards Institute (CLSI).

Office Bacteriology

Many office practices perform rapid antigen testing for detecting group A streptococcal pharyngitis. Sensitivity depends on the type of kit used and on the concentration of streptococci present in the sample. Practitioners should investigate the specific tests available to them. As many as 30% of test results may be false-negative; therefore, all negative results should be confirmed by culture.

Other microbiologic tests may be performed in the office setting, provided the site is certified as meeting appropriate quality-assurance standards specified by the Clinical Laboratory Improvement Amendments (CLIA) of 1988. These include procedures listed under the category of “provider-performed microscopy” such as wet mounts, potassium hydroxide (KOH) preparations, pinworm examinations, fecal leukocyte examinations, and urine sediment analysis. Office laboratories licensed to perform waived tests are limited to performing these tests and avoid having to undergo inspections and proficiency testing, although they are still subject to CLIA certification requirements specific to these tests. Gram staining, culture inoculation, and isolation of bacteria are considered moderately to highly complex tests under CLIA specifications. Any office laboratory performing Gram stains or cultures must comply with the same requirements and inspections for quality assurance, proficiency testing, and personnel requirements as fully licensed microbiology laboratories.

Laboratory Diagnosis of Viral Infections

Specimens for viral diagnosis are selected on the basis of knowledge of the site that is most likely to yield the suspected pathogen. Specimens should be collected early in the course of infection when viral shedding is maximal. Fluids and respiratory secretions should be collected in sterile containers and promptly delivered to the laboratory. Swabs should be rubbed vigorously against mucosal or skin surfaces to obtain as much cellular material as possible and sent in viral transport media that contain antibiotics to inhibit bacterial growth. Rectal swabs should not be heavily covered with feces, because the antibiotics present in viral transport media may be insufficient to kill a large inoculum of bacteria. All specimens should be transported on ice. Freezing specimens can result in a significant decrease in culture sensitivity. Consultation with the laboratory is advised for any unusual specimens or suspected pathogens.

Laboratory diagnosis of viral infections may be by electron microscopy, antigen detection, virus isolation in culture, serologic testing, or detection of virus genomes by molecular biology techniques. Serologic and molecular tests are the mainstay of diagnosis of viruses such as HIV and Epstein-Barr virus (EBV) and increasingly for many respiratory viruses.

Rapid Antigen Tests

Immunofluorescent-antibody (IFA) techniques or other methods such as EIA that use antibodies to detect viral antigens directly in clinical specimens permit rapid identification of viruses. Smears of cellular material from respiratory secretions stained by immunologic reagents can identify the antigens of respiratory syncytial virus (RSV), adenovirus, influenza virus, and parainfluenza virus within 2-3 hr after the specimen is received. In comparison with isolation in cell culture, sensitivity of IFA is approximately 80-90% for the diagnosis of RSV and parainfluenza virus type 3 in reference laboratories; the sensitivity of IFA for influenza viruses and adenoviruses is considerably lower. Novel influenza strains such as the one responsible for H1N1 pandemic influenza are poorly detected by IFA and require molecular tests.

Sensitive IFA staining techniques are also commercially available for identifying varicella-zoster virus (VZV) and herpes simplex virus (HSV). These specific methods have supplanted the Tzanck smear for multinucleated giant cells characteristic of VZV or HSV infections. A method for detecting cytomegalovirus (CMV) antigen in blood of immunocompromised patients is also available. IFA is not useful for detecting virus in specimens that do not contain an adequate number of infected cells. When possible, an accompanying specimen for virus isolation usually is advisable.

In addition to providing rapid diagnosis, antigen-detection EIA tests are commonly used for the diagnosis of viruses that are difficult to culture, such as rotavirus and hepatitis B virus.

Isolation and Identification

Viruses require living cells for propagation; the cells used most often are human- or animal-derived tissue culture monolayers, such as human embryonic lung fibroblasts or monkey kidney cells. In vivo methods for isolation are sometimes necessary (suckling mice inoculation for culture of arboviruses and rabies virus). Because viruses require various cell culture systems for isolation, it is important for clinicians to provide relevant clinical information to the laboratory to facilitate selection of appropriate cell lines.

Viral growth in susceptible cell cultures can be detected in several ways. Many viruses produce a characteristic cytopathic effect (CPE) that is visible by light microscopy under low magnification. For example, RSV and HSV produce multinucleated giant cells and syncytia formation. Other viruses (e.g., influenza and mumps) can be detected by hemadsorption, because hemagglutinins on infected cell membranes permit adherence of erythrocytes to infected cells. The most reliable confirmatory method for viral detection in cell culture involves fluorescein- or enzyme-labeled monoclonal antibody staining of infected cell monolayers.

An important technical improvement in respiratory viral cultures is the development of an engineered tissue monolayer (R-mix) for rapid shell vial detection of influenza A and B, RSV, parainfluenza 1-3, and adenoviruses. Respiratory shell vial cultures have turnaround times of 2 days compared with 2-3 wk for conventional cultures and are most useful for rapid diagnosis of influenza infections where sensitivity of IFA is low and rapid detection can result in early institution of appropriate antiviral therapy.

Laboratory Diagnosis of Parasitic Infections

Most parasites are detected by microscopic examination of clinical specimens. Plasmodium and Babesia can be detected in stained blood smears, Leishmania can be detected in stained bone marrow smears, and helminth eggs and Entamoeba histolytica and Giardia lamblia cysts and trophozoites can be detected in stained fecal smears (see Table 164-1). Serologic tests are important in documenting exposure to certain parasites that are difficult to demonstrate in clinical specimens, such as Trichinella and Toxoplasma. Serologic testing also has a role in the diagnosis of intestinal strongyloidiasis, given the insensitivity of stool examinations.

Fecal specimens should not be contaminated with water or urine, because water can contain free-living organisms that can be confused with human parasites, and urine can destroy motile organisms. Mineral oil, barium, and bismuth interfere with the detection of parasites, and specimen collection should be delayed for 7-10 days after ingestion of these substances. Because Giardia and many worm eggs are shed intermittently into feces, a minimum of three specimens are required for an adequate examination. It is recommended that the three specimens be collected on separate days, preferably on alternate days. Because many protozoan parasites are easily destroyed, collection kits with stool preservatives should be used if delay between time of specimen collection and transport to the laboratory is anticipated.

Ova and parasite examination of fecal specimens includes a wet mount (to detect motile organisms if fresh stool is received), concentration (to improve yield), and permanent staining, such as trichrome, for microscopic examination. These techniques can miss parasites such as Cryptosporidium, Cyclospora, and microsporidia. Cryptosporidium and Cyclospora are detected by modified acid-fast stain and microsporidia by a modification of the trichrome stain. The laboratory should be alerted if these parasites are suspected. Detection of certain parasites, especially Giardia, E. histolytica, and Cryptosporidium, can be simplified by using sensitive EIA antigen detection tests. Rapid antigen detection (dipstick) tests for Plasmodium falciparum and P. vivax such as those based on detection of Plasmodium histidine-rich protein are also available with sensitivities and specificities comparable to expert microscopy. These tests are particularly useful in areas where trained laboratory personnel are not available.

Serologic Diagnosis

Serologic tests are primarily used in the diagnosis of infectious agents that are difficult to culture in vitro or detect by direct examination, such as Bartonella, Legionella, Borrelia (Lyme), Treponema pallidum, Mycoplasma, Rickettsia, Ehrlichia, some viruses (HIV, EBV, hepatitis A and B viruses), and parasites (Toxoplasma, Trichinella).

Antibody tests may be specific for immunoglobulin G (IgG) or M (IgM) or can measure antibody response regardless of immunoglobulin class. The IgM response occurs earlier in the illness, generally peaking at 7-10 days after infection, and usually disappears within a few weeks, but for some infections (e.g., hepatitis A) it can persist for months. The IgG response peaks at 4-6 wk and usually persists for life. Because the IgM response is transient, the presence of IgM antibody in most cases correlates with recent infection; therefore, a single positive serum specimen is considered diagnostic. Methods for IgM antibody detection are difficult to standardize, however, and false-positive results commonly occur with some tests. The presence of IgG antibody can indicate new seroconversion or past exposure to the pathogen. To confirm a new infection using IgG testing, it is essential to demonstrate either seroconversion or a rising IgG titer. A fourfold increase in a convalescent titer obtained 2-3 wk after the acute titer is considered diagnostic in most situations. However, for some infections (e.g., Bartonella, Legionella, and rickettsiae) a single positive IgG titer is sufficient for diagnosis.

Serologic diagnosis of Lyme disease remains problematic because of lack of specificity of the commercial enzyme immunoassays. A confirmatory immunoblot (Western blot) is required for all positive and equivocal EIA results for Lyme disease. For early Lyme disease, both IgG and IgM antibody positivity (“bands”) are required for confirmation, whereas for late disease, only an IgG immunoblot assay should be performed, because false-positive reactions with IgM immunoblot assays are common. Ordering serologic tests for children with nonspecific complaints such as arthralgia or fatigue is unnecessary and creates needless alarm, because invariably these represent false-positive reactions.

Several rapid enzyme-linked immunospot (ELISPOT) assays that detect production of interferon-γ (IFN-γ) by tuberculosis-specific lymphocytes in the patients’ blood. IFN-γ release assays (IGRAs) are now FDA approved for diagnosis of tuberculosis disease or latent infection. Lack of cross-reactivity with BCG and higher specificity make IGRAs useful in BCG-immunized children. IGRAs can also be used as alternatives to tuberculin skin testing (TST) in immunocompetent children ≥5 yr of age. As with TST, IGRAs cannot distinguish between tuberculosis disease and latent infection, and a negative test does not rule out the possibility of disease in children with high clinical suspicion. Guidelines for use of IGRAs have been developed by the Centers for Disease Control and Prevention (CDC).

Molecular Diagnostic Techniques

Molecular diagnostic techniques are most useful for detecting and identifying pathogens for which culture and serologic tests are difficult, slow, or not available. Molecular diagnostic testing for infectious agents is common and has revolutionized clinical microbiology with full-scale automation, high sensitivities and specificities, and rapid reporting of results. Two of the widely used techniques in clinical microbiology are DNA probes for direct detection and nucleic acid amplification using polymerase chain reaction (PCR). Microbiologic applications of techniques such as miniaturized DNA chip technology and microarrays based on the ability of DNA to find and spontaneously bind to its complementary sequences (for example, mycobacterial DNA in a patient’s blood) are also being developed.

DNA probes detect or identify organisms by hybridization of the probe to complementary sequences in DNA or ribosomal RNA. The principal use of DNA probe technology remains rapid identification of organisms that already have been isolated in culture but require additional time-consuming or complex confirmation procedures. Probes for mycobacterial species can rapidly distinguish M. tuberculosis from M. avium complex growing in broth cultures. Detection of pathogen nucleic acid directly in clinical specimens is also possible but requires the presence of relatively large numbers of organisms in the specimen. Commercially available probes for direct detection of pathogen from specimens include combination probes for detection of C. trachomatis and gonococci from genitourinary specimens.

The high sensitivity and specificity of PCR amplification make this the method of choice for direct detection of microbial nucleic acid from clinical specimens. The PCR method is based on the ability of thermostable DNA or RNA polymerase to copy targeted gene sequences using complementary nucleotides as primers to amplify a conserved region of the genome. The reaction takes place in a thermal cycler. Each cycle of the reaction theoretically doubles the amount of target nucleic acid, resulting in more than a million-fold amplification after 30 cycles of PCR. The greatest impact of PCR is in clinical virology and mycobacteriology, where conventional methods are slow and insensitive.

The number of pathogens that can be detected by PCR is increasing rapidly. PCR tests are available using commercial reagents for many respiratory viruses, HIV, hepatitis B and C viruses, CMV, and C. trachomatis. FDA-approved PCR tests are also available for M. tuberculosis for use on respiratory specimens. These tests have lower sensitivity for gastric aspirates, CSF, and tissue specimens. Multiplex tests such as the Hexaplex assay (Prodesse, Milwaukee, WI) for detecting seven common respiratory viral pathogens in children are also commercially available. Experimental PCR protocols for detecting Bartonella, B. pertussis, Legionella, M. pneumoniae, Chlamydia pneumoniae, and HSV are available in some reference laboratories. PCR primers for the highly conserved regions of the bacterial 16S ribosomal RNA as a direct test or combined with an initial culture can hasten an accurate (universal primer) diagnosis of serious bacterial infections.

Specimens for PCR should be sent in separate sterile containers and rapidly transported. Traditional PCR methods are technically complex and labor intensive. False-positive reactions are a major problem, because the extreme sensitivity of the assay can lead to amplification of target nucleic acid from extraneous sources or from crossover contamination from other positive specimens. Technical improvements in thermocycler equipment (quantitative real-time PCR) using sealed tubes incorporating fluorescent probes added to the PCR mix to confirm and quantify PCR products as they are generated have significantly reduced processing times and postamplification contamination. Turnaround times of 2-4 hr from receipt of specimen are now possible. Real-time PCR has been used for rapid detection of many pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococcal colonization, and respiratory viruses. Clinically relevant diagnostic PCR testing should be performed only in reference laboratories using adequate quality control measures.

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