Diagnosis of infection and assessment of host defence mechanisms

31 Diagnosis of infection and assessment of host defence mechanisms

Aims of the clinical microbiology laboratory

The aims of the microbiology laboratory are:

Identification is achieved by detecting the microorganism or its products or the patient’s immune response

Laboratory tests are carried out:

While there are different protocols for different specimens (e.g., urine, faeces, genital tract, blood, etc.), the tests fall into three main categories:

1. Identification of microorganisms by isolation and culture. Microorganisms may grow in artificial media or, in the case of viruses, in cell cultures. In some instances, quantification is important (e.g. more than 105 bacteria/mL of urine is indicative of infection whereas lower numbers are not; see Ch. 20). Once an organism has been isolated in culture, its susceptibility to antimicrobial agents can be determined.

2. Identification of a specific microbial gene or product. Non-cultural techniques that do not depend upon the growth and multiplication of microorganisms to detect microorganisms have the potential to yield more rapid results. These techniques include the detection of structural components of the cell (e.g. cell wall antigens) and extracellular products (e.g. toxins). Alternatively, molecular approaches are increasingly available such as the detection of specific gene sequences in clinical specimens using DNA probes or the polymerase chain reaction (PCR; see below). They are potentially applicable to all microorganisms, but antimicrobial susceptibilities cannot be determined without culture (although the presence of resistance genes may be detectable by specific probes).

3. Detection of specific antibodies to a pathogen. This is especially important when the pathogen cannot be cultivated in laboratory media (e.g. Treponema pallidum, many viruses) or when culture would be particularly hazardous to laboratory staff (e.g. culture of Francisella tularensis, the cause of tularaemia, or the fungus Coccidioides immitis). Detection of IgM and/or IgG antibodies in a single serum collected during the acute phase of illness can be helpful in diagnosis of, for example, rubella by specific IgM, hepatitis A by IgM and hepatitis B by HepB surface antigen, or in rare diseases such as Lassa fever. The classic diagnostic method is by detection of a rise (fourfold or greater) in antibody titre between ‘paired’ sera, collected in the acute phase of an infection (5–7    days after onset of symptoms) and in convalescence (after 3–4    weeks). Such tests therefore tend to result in a delayed or retrospective diagnosis and are therefore of limited help for clinical management.

Specimen processing

Specimen handling and interpretation of results is based upon a knowledge of normal flora and contaminants

Specimens intended for cultivation of microorganisms can be divided into two types:

A thorough knowledge of the microorganisms normally isolated from specimens from non-sterile sites, and the common contaminants of specimens collected from sterile sites, is important to ensure that specimens are properly handled and the results are correctly interpreted. Some specimens from sites that should be sterile (e.g. bladder urine, sputum from the lower respiratory tract) are usually collected after passage through orifices that have a normal flora, which may contaminate the specimens. This needs to be considered when interpreting the culture results of these specimens.

In an ideal world, each specimen arriving in the laboratory would be considered in turn together with the information provided about the patient on the request form so that the microbiologist could assess the pathogens likely to be present and devise an ‘individualized’ processing plan. However, in reality, this approach is not practicable because of constraints on time and money. Thus, specimens tend to be processed by type (e.g. urine, blood, faeces) and the microbiologist looks for easily cultivated pathogens known to be associated with each sample type. However, if the laboratory is provided with suitable information, such as a statement of possible aetiology, more fastidious or unusual pathogens can be sought and relevant antibiotic susceptibilities assessed. To obtain a test result that correctly identifies the infection, it is important to collect an appropriate specimen, to use the appropriate transport conditions and to deliver specimens rapidly to the laboratory. These conditions all affect the accuracy of the laboratory report, and therefore its value to the clinician and ultimately to the patient. Key points to remember about specimen collection are summarized in Box 31.2.

Non-cultural techniques for the laboratory diagnosis of infection


Microscopy is an important first step in the examination of specimens

Microscopy plays a fundamental role in microbiology. Although microorganisms show a wide range in size (see Ch. 1) they are too small to be seen individually by the naked eye, and therefore a microscope is an essential tool in microbiology. The various types of microscopy are summarized in Figure 31.2. The light microscope magnifies objects and therefore improves the resolving power of the naked eye from about 100 000    nm (0.1    mm) to 200    nm; the electron microscope can improve this to 0.1 to 1.0    nm.

Light microscopy

Acid-fast stains are used to detect mycobacteria

Some organisms, particularly mycobacteria, which have waxy cell walls, do not readily take up the Gram stain. To demonstrate their presence, special staining techniques are used which rely on the ability of such organisms to retain the stain in the presence of ‘decolourizing’ agents such as acid and alcohol. The Ziehl–Neelsen stain (see Fig. 19.20) is a classic differential staining procedure that uses heat to drive the fuchsin stain into the cells; mycobacteria stained with fuchsin withstand decolourization with acid and alcohol and are therefore known as ‘acid-’ and ‘alcohol-fast’, whereas other bacteria lose the stain after acid and alcohol treatment. Alternatively, many laboratories use the fluorescent dye auramine, which has a strong affinity for the waxy cell wall of mycobacteria, to demonstrate these organisms by fluorescence microscopy (Fig. 31.4).

Electron microscopy

Detection of microbial antigens in specimens

Detection of specific microbial antigens can be a more rapid method for detecting the presence of an organism than attempting to grow and identify the microbe. The methods include:

They are summarized in Box 31.3. Detection of microbial genes using DNA probes is discussed later in this chapter.

Jul 9, 2017 | Posted by in MICROBIOLOGY | Comments Off on Diagnosis of infection and assessment of host defence mechanisms

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