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INTRODUCTION

The laboratory diagnosis of infection requires the demonstration—either direct or indirect—of viral, bacterial, fungal, or parasitic agents in tissues, fluids, or excreta of the host. The traditional detection methods of microscopy and phenotypic characterization are time-consuming and are increasingly being replaced by nucleic acid probe assays.

MICROSCOPY

  • Wet mounts: the simplest method for microscopic evaluation and useful for certain large and/or motile organisms. For example, combining wet mounts with dark-field illumination permits detection of spirochetes in genital lesions or of Borrelia and Leptospira in blood.

    • – Fungal elements may be identified in skin scrapings with 10% KOH wet mount preparations.

    • – Some wet mounts use staining to enhance detection and morphologic identification—e.g., india ink for visualization of encapsulated cryptococci in CSF and lactophenol cotton blue for morphologic identification of fungal elements.

  • Stains: Staining techniques permit organisms to be seen more clearly.

    • Gram's stain: differentiates between organisms with thick peptidoglycan cell walls (gram-positive) and those with thin peptidoglycan cell walls and outer membranes that can be dissolved with alcohol or acetone (gram-negative).

      • This stain is particularly useful for examining sputum samples for PMNs and bacteria. The presence of >10 epithelial cells per low-power field and multiple bacterial types suggests contamination with oral flora.

      • In normally sterile fluids (e.g., CSF, pleural and joint fluid), the detection of bacteria suggests an infectious etiology (Fig. 85-1) and correlates with the presence of >104 bacteria/mL.

      • Sensitivity is increased by centrifugation of the sample.

    • Acid-fast stains: identify organisms that retain carbol fuchsin dye after acid/organic solvation (e.g., Mycobacterium spp.). Modification of this procedure permits detection of weakly acid-fast organisms (e.g., Nocardia).

    • Immunofluorescent stains: utilize antibodies—either labeled directly with a fluorescent compound or detected indirectly by a secondary immunofluorescent antibody—to detect viral antigens (e.g., CMV, HSV, and respiratory viruses) within cultured cells and difficult-to-grow bacteria (e.g., Legionella pneumophila).

MACROSCOPIC ANTIGEN DETECTION

  • Latex agglutination assays and EIAs are rapid and inexpensive tests that identify bacteria, viruses, or extracellular bacterial toxins by means of their protein or polysaccharide antigens.

  • The assays may be performed either directly on clinical specimens or after growth of the organisms in the laboratory.

CULTURE

  • The success of efforts to culture a specific pathogen often depends on use of appropriate collection and transport procedures in conjunction with a laboratory-processing algorithm suitable for the specimen. Instructions for collection and transport are listed in Table 85-1.

  • Bacterial isolation relies on the use of artificial media that support bacterial growth in vitro. Once bacteria are isolated, different methods are used to characterize specific isolates (e.g., phenotyping based on enzymatic and metabolic functions, gas-liquid chromatography, nucleic acid tests).

  • Viruses are grown on a monolayer of cultured cells ...

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