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77 DIAGNOSIS

77 DIAGNOSIS
Harrison’s Manual of Medicine

77

DIAGNOSIS

Direct Detection
Detection by Culture
Detection by Serologic Methods
Detection by Nucleic Acid Probes
Bibliography
The laboratory diagnosis of infection requires the demonstration, either directly or indirectly, of viral, bacterial, mycotic, or parasitic agents of disease in tissues, fluids, or excreta of the host. The cornerstone for the diagnosis of parasitic infections (Table 77-1), as of many other infections, is a thorough history of the pt’s illness, including occupation, recreation, and travel. The traditional detection methods of microscopy and/or culture are increasingly being complemented by more rapid and sensitive techniques, including serology, nucleic acid probing, and polymerase chain reaction (PCR).

Table 77-1 Diagnosis of Some Common Parasitic Infections

DIRECT DETECTION
MICROSCOPY   Wet Mounts   The wet mount is the simplest method for microscopic evaluation, involving no fixation of the specimen prior to examination. In general, it is used for certain large and/or motile organisms that can be visualized without staining. Wet mounts of duodenal aspirates may show pathogenic protozoans (e.g., giardial trophozoites), while mounts of fresh stool may reveal protozoans or helminths (e.g., amebic cysts, Strongyloides larvae, ascarid or schistosome eggs). Mounts of fresh blood may reveal microfilariae (in brugian or bancroftian filariasis or loiasis) or spirochetes (in relapsing fever). Motile trichomonads may be found in cervical secretions. Wet mounts with dark-field illumination are used to detect Treponema in spirochetal genital lesions and to reveal Borrelia or Leptospira in blood. To detect fungal elements in skin scrapings or hair, 10% KOH preparations may be used. For certain wet- mount applications, staining is used to enhance detection or visualization of morphologic elements (e.g., India ink to visualize encapsulated cryptococci in CSF, lactophenol cotton blue for fungal speciation).
Gram’s Stain   Gram’s stain enhances detection of bacteria and PMNs and differentiates bacteria with thick peptidoglycan cell walls (gram-positive; purple) from those with alcohol- or acetone-labile outer membranes (gram-negative; pink). In a properly decolorized slide, the nuclei of PMNs appear pink. Purulent respiratory secretions have >25 PMNs and <10 epithelial cells per low-power field; the presence of >10 epithelial cells per low-power field reflects contamination of the specimen by saliva. A bacterial content of >104/mL in a specimen from a normally sterile site should be detectable by Gram’s stain. Specimens may be concentrated by centrifugation to promote the detection of rare organisms.
Acid-Fast Stain   The acid-fast stains detect organisms, such as Mycobacterium spp., that are capable of retaining carbol fuchsin dye after acid/organic solvation (e.g., 3% HCl in 95% ethanol). The modified acid-fast stains detect weakly acid-fast organisms, such as Nocardia and Cryptosporidium, which retain the dye on treatment with dilute acid (e.g., 1% HCl) but not acid-alcohol. Acid-fast organisms appear pink or red against the blue background of the counterstain. Because mycobacteria may be sparse in clinical specimens, the more sensitive auramine-rhodamine combination fluorescent dye technique was developed.
Other Stains for Light Microscopy   Giemsa or Wright’s stain of peripheral blood may reveal certain bacteria and intra- or extracellular parasites (e.g., Borrelia recurrentis, Plasmodium, Babesia, or Trypanosoma). Other commonly used stains include toluidine blue (for Pneumocystis carinii) and methenamine silver (for P. carinii and for fungal hyphae in tissue sections).
Immunofluorescent Stains   Immunofluorescent stains can be used to detect viruses within cultured cells or tissue specimens (e.g., herpesviruses, rabies virus) or to reveal difficult-to-grow bacterial agents within clinical specimens (e.g., Legionella pneumophila). Direct immunofluorescent antibody (DFA) stains use antibodies directed at the agent of interest coupled to a fluorescent compound such as fluorescein, which directly labels the target. Indirect immunofluorescent antibody (IFA) stains use an unlabeled primary antibody to the target antigen followed by a labeled secondary antibody to the primary antibody. Since a single primary antibody molecule is bound by many secondary antibody molecules, the fluorescent signal is amplified with the indirect approach. In either case, the stained specimen is examined under UV light, and visible light is emitted.
MACROSCOPIC ANTIGEN DETECTION   Latex agglutination assays and enzyme immunoassays (EIAs) are rapid and inexpensive methods for identifying bacteria, viruses, or extracellular bacterial toxins by means of their protein or polysaccharide antigens. These assays can be performed either directly on clinical specimens or after growth of organisms in the laboratory. Conditions that may be diagnosed by these means include cryptococcosis, histoplasmosis, salmonellosis, shigellosis, and infections due to Clostridium difficile toxins A and B.
DETECTION BY CULTURE
The success or failure of efforts to culture bacterial, mycotic, or viral pathogens depends critically on the nature of the sample provided, the means by which it is collected and transported, and the use of a laboratory processing algorithm suitable for the specific sample. Table 77-2 lists procedures for collection and transport of common clinical specimens. Physicians in doubt about the procedure appropriate for a particular situation should seek advice from the microbiology laboratory before obtaining the specimen.

Table 77-2 Instructions for Collection and Transport of Specimens for Culture

DETECTION BY SEROLOGIC METHODS
Measurement of serum antibody to a specific agent provides indirect evidence of past or current infection. Serologic methods are used to detect many viral infections and have applications in other areas of microbiologic diagnosis as well. The value of antibody assays in parasitic diagnosis is limited by slow development and the inability to distinguish between past and present infection. However, the restricted geographic distribution of many parasites increases the diagnostic usefulness of these assays in travelers from industrialized countries. Detection systems include agglutination reactions, immunofluorescence, EIA, hemagglutination inhibition, and CF. Serologic methods may be used to establish the existence of immunity by documenting that antibody quantity exceeds the protective level (e.g., for rubella, rubeola, or varicella-zoster virus) or to detect current infection by demonstrating a rise in antibody titer between acute- and convalescent-phase samples collected 10–14 d apart (e.g., for arboviral infections, brucellosis, legionellosis, or ehrlichiosis).
DETECTION BY NUCLEIC ACID PROBES
Techniques to detect pathogen-specific DNA or RNA sequences in clinical specimens have become powerful tools for microbiologic diagnosis. All such techniques capitalize on the great specificity of Watson-Crick base pairing. Probes are available to detect a number of pathogens directly in clinical specimens (e.g., L. pneumophila, Chlamydia trachomatis, Neisseria gonorrhoeae, group A Streptococcus, Gardnerella vaginalis, Trichomonas vaginalis, Mycoplasma hominis, and Giardia lamblia). Other probes are available for confirmation of culture results (e.g., those for Mycobacterium and Salmonella spp.). The sensitivity and specificity of probe assays are comparable to those of culture or EIA. Nucleic acid amplification strategies have also entered the clinical arena. PCR is the best known of these and is far more sensitive than traditional detection methods. It is, however, susceptible to false-positives from even low levels of contaminaton. Amplification assays are currently available to detect Mycobacterium tuberculosis, N. gonorrhoeae, M. hominis, and C. trachomatis.
Bibliography

For a more detailed discussion, see Onderdonk AB: Laboratory Diagnosis of Infectious Diseases, Chap. 121, p. 775; and Davis CE: Laboratory Diagnosis of Parasitic Infections, Chap. 211, p. 1186, in HPIM-15.

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