Histologic-type stains are used daily in the microbiology laboratory; most often Gram stain and acid-fast stain (Ziehl-Neelsen or its modifications). Both of these stains detect certain bacteria and to some extent help identify them. Other histologic stains perform the same purpose in tissue slide examination; the most common are paraaminosalycylic acid (PAS) and silver stains for fungi. Immunohistologic stains containing antibody against organism antigen can be used on smears or histologic slides to identify organisms, although this is not frequently done.
Category: Bacterial Infectious Diseases (Including Chlamydia, Mycoplasma, and Legionella Infections)
Bacterial Infectious Diseases (Including Chlamydia, Mycoplasma, and Legionella Infections)
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Serologic Tests
In many cases, direct detection methods are not possible, are difficult and expensive, are unreliable, or are attempted with negative results. Serologic tests attempt to detect antibodies formed against antigens of an organism being searched for. The majority of organisms have a reasonably predictable antibody response. IgM-type antibodies appear first, most often in 7-14 days (sometimes later), generally reaching peak titer about 2 weeks later, then falling, and becoming nondetectable about 6 months later (usual range, 4-8 months, although sometimes longer). IgG-type antibodies typically appear about 2 weeks after initial appearance of IgM antibodies, peak about 2 weeks later, and typically persist for years (although some eventually disappear and others may slowly decrease in titer and persist at low titers). If antibodies to a particular organism are rare in a community, a single significantly elevated result can be at least presumptively diagnostic. However, most often it requires two serum specimens, one obtained immediately and the other 2-3 weeks later, hoping to obtain a fourfold (2-tube serial dilution) rise in titer in order to prove current or recent infection. Potential problems include circumstances in which serologic tests for certain organisms are not available, tests ordered to detect one organism when another is the culprit, patients who fail to produce detectable antibody, patient antibody formation that varies in time from the usual pattern, serum specimens obtained before antibody rise or after antibody becomes nondetectable, and specimens obtained after antibody has peaked so that past infection cannot be differentiated from current infection. In the last of these circumstances, presence of IgM antibody in high titer would suggest current or relatively recent infection. Finally, there is substantial variation in homemade or commercially available test kits, both in ability to detect the desired antibodies (sensitivity) and the number of false positive results obtained (specificity).
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Direct Methods of Bacterial Detection
Culture. This is the classic definitive method for detection and identification and will be discussed later in more detail. The major drawback is time; it usually takes 1 full day to grow the organism and then part or all of 1 day to identify it. It may take an additional day to isolate it before identification if there is a mixture of organisms. Some organisms take longer than 1 day to grow. There is always a certain percentage of false negative results (sometimes a large percentage) due to various factors, both clinical and technical. Several major difficulties are suppressive effects of antibiotic therapy on bacterial growth (even though clinical cure is not achieved); specimen not obtained from its best area (sampling error), inadequate or inappropriate specimens obtained, or faulty specimen transport to the laboratory; and differences in the way any individual laboratory processes the specimen compared to a research laboratory.
Immunologic methods. Immunologic methods (immunoassay) depend on antigen-antibody reaction, either test antibody binding to patient antigen or test antigen attachment to patient antibody. There also must be a readout or indicator system to show that the reaction has taken place and to quantify the amount of patient antigen or antibody. The indicator can be a radioactive molecule (radioimmunoassay [RIA]), a fluorescent molecule (fluorescent immunoassay [FIA]), a molecule with an attached enzyme that can participate in a biochemical color reaction (enzyme-linked immunoassay [ELISA or EIA]), or some other method, such as an inert particle coated with antigen or antibody that produces particle agglutination as the endpoint of the reaction (e.g., latex particle agglutination [LA]). There can be a single-reagent antibody or antigen that captures the antigen and a second antibody that contains the readout molecule and that attaches to the captured patient antigen (“sandwich” immunoassay). The antibody used may be produced in animals and is not completely specific for the selected antigen (polyclonal antibody); or an antibody may be produced that is specific for an antigen or a particular receptor (epotope) on the antigen (monoclonal antibody). Considerably simplified, monoclonal antibodies currently are most often produced by injecting the antigen into a mouse, waiting until the mouse produces antibody against the antigen, obtaining samples of the mouse spleen and culturing different lymphocytes until one is found that produces a specific antibody, then incubating the mouse lymphocyte with a myeloma cell and providing an environment (e.g., polyethylene glycol) that causes the two cells to stick together and then fuse into one hybrid cell. The myeloma inheritance causes the cell (and its offspring) to rapidly reproduce for long periods of time, while the mouse spleen inheritance results in continued specific (monoclonal) antibody production. Some of the immunologic methods are capable of accuracy that is equivalent to culture (e.g., fluorescent anti- body method for Corynebacterium diphtheriae); others are less reliable, depending on the technique and the particular kit manufacturer. All antibodies do not behave the same, even under the same conditions.
Nucleic acid probe (DNA probe). Greatly simplified, this technique attempts to construct a nucleic acid sequence (the “probe”) that matches a sequence in the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of the organism to be detected. This sequence or probe is incorporated (or grafted) into a larger nucleic acid molecule, usually a single strand of DNA (although a strand of RNA could be used) that can be tagged with an indicator system (radioisotope or biochemical reaction). Then the specimen to be tested is prepared for analysis. If the target molecule is DNA, since DNA usually exists as a double-stranded molecule, the target molecule DNA double strands are first separated into single strands by various means, and then the test DNA single strands containing the probe sequence are introduced. If the probe sequence matches a sequence in the target, the probe hybridizes with the target DNA (combines with the target single strand) to again form a double strand. If the target molecule is RNA, RNA exists in living cells in single-strand state, so that the DNA single-strand test molecule containing the RNA probe area can bypass the strand separation step and hybridize directly with an RNA single strand (instead of another DNA single strand) if the probe area matches a nucleic acid sequence in the target RNA strand. After incubation, nonhybridized (nonattached) test probe-carrying single strands are washed away and the indicator system is used to demonstrate whether any probe remains combined to target molecules. In some ways this technique is similar to direct immunologic detection methods. Advantages of nucleic acid probe systems are much greater sensitivity than current antibody systems; specificity that can be varied to the genus, species, or even strain level; theoretical possibility of use for any organism in any clinical specimen; and same-day results. The major disadvantage thus far is relatively high cost of the test when performed on a single specimen basis. -
Bacterial Infectious Diseases (Including Chlamydia, Mycoplasma, and Legionella Infections)
Proper therapy for infectious disease requires knowledge of the etiologic agent. This knowledge can be gained in two ways: directly, either by isolating and identifying the organism in culture or by some method (e.g., fluorescent antibody or nucleic acid probe) that permits specific visual detection of the organism in clinical material; or indirectly, by using serologic tests that demonstrate antibodies against an organism in the patient’s blood. Certain problems arise in the interpretation of direct methods or serologic test results. If results of the direct method are positive, one must determine whether the organism reported has clinical significance. It may be a fortuitous contaminant, an ordinarily nonpathogenic species that has become infectious under the prevailing circumstances, or a recognized pathogen that is a normal inhabitant of the area and whose presence may be entirely harmless. If results of the direct method are negative, one must consider the possibility of having missed the diagnosis because the laboratory was not given a proper specimen, because the specimen was obtained at an unfavorable moment during the disease, or because of laboratory technical problems (and, if culture is involved, because special culture techniques or media might have been needed). Serologic tests that detect antibodies also have drawbacks in bacterial diseases, since it takes 7-10 days after infection (sometimes longer) to develop detectable antibodies, and it is often necessary to demonstrate either a very high titer or a fourfold rising titer of antibodies to differentiate recent from old infection. Therefore, to evaluate laboratory data, the clinical situation has at least as much importance as laboratory methodology. It seems desirable to provide a brief survey of the major infectious agents, the diseases and circumstances in which they are most often found, and the conditions under which the appearance of these organisms may be confusing. There is an element of classification because of the way laboratory reports are usually worded. Techniques of diagnosis in the area of infectious disease are discussed in this chapter in relation to specific organisms, specific clinical situations, or general laboratory methods.