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INTRODUCTION

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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. Clinical microbiology laboratories are responsible for processing these specimens and also for determining the antibiotic susceptibility of bacterial and fungal pathogens. Traditionally, detection of pathogenic agents has relied largely on either the microscopic visualization of pathogens in clinical material or the growth of microorganisms in the laboratory. Identification generally is based on phenotypic characteristics such as fermentation profiles for bacteria, cytopathic effects in tissue culture for viral agents, and microscopic morphology for fungi and parasites. These techniques are reliable but are often time-consuming. Increasingly, the use of nucleic acid probes is becoming a standard method for detection, quantitation, and/or identification in the clinical microbiology laboratory, gradually replacing phenotypic characterization and microscopic visualization methods. This chapter discusses general concepts of diagnostic testing, with an emphasis on detection of bacteria. Detection of viral, fungal, and parasitic pathogens is discussed in greater detail in separate chapters (see Chaps. 86, 110, and 120, respectively).

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DETECTION METHODS

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Reappraisal of the methods employed in the clinical microbiology laboratory has led to the development of strategies for detection of pathogenic agents through nonvisual biologic signal detection systems. A biologic signal is generated by detection of a material that can be reproducibly differentiated from other substances present in the sample. Key issues in the use of a biologic signal are distinguishing it from background noise and translating it into meaningful information. Examples of useful materials for detection of biologic signals applicable to clinical microbiology include structural components of bacteria, fungi, and viruses; specific antigens; metabolic end products; unique DNA or RNA base sequences; enzymes; toxins or other proteins; and surface polysaccharides.

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A detector is used to sense a signal and discriminate between that signal and background noise. Detection systems range from the trained eyes of a technologist assessing morphologic variations to electronic instruments such as gas-liquid chromatographs or mass spectrometers. The sensitivity with which signals can be detected varies widely. It is essential to use a detection system that discerns small amounts of signal even when biologic background noise is present—i.e., that is both sensitive and specific. Common detection systems include immunofluorescence; chemiluminescence for DNA/RNA probes; flame ionization detection of short- or long-chain fatty acids; and detection of substrate utilization or end-product formation as color changes, of enzyme activity as a change in light absorbance, of turbidity changes as a measure of growth, of cytopathic effects in cell lines, and of particle agglutination as a measure of antigen presence.

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Amplification enhances the sensitivity with which weak signals can be detected. The most common microbiologic amplification technique is growth of a single bacterium into a discrete, visible colony on an agar plate or into a suspension containing many identical organisms. The advantage of growth as an amplification method is ...

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