Read More
Date: 15-2-2016
10412
Date: 16-2-2016
1956
Date: 15-2-2016
1149
|
Principle of diagnostic medical microbiology
Introduction
Diagnostic medical microbiology is concerned with the etiologic diagnosis of infection. Laboratory procedures used in the diagnosis of infectious disease in humans include the following:
(1) Morphologic identification of the agent in stains of specimens or sections of tissues (light and electron microscopy).
(2) Culture isolation and identification of the agent.
(3) Detection of antigen from the agent by immunologic assay (latex agglutination, EIA, etc) or by fluorescein-labeled (or peroxidase-labeled) antibody stains.
(4) DNA-DNA or DNA-RNA hybridization to detect pathogen-specific genes in patients' specimens.
(5) Detection and amplification of organism nucleic acid in patients' specimens.
(6) Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.
In the field of infectious diseases, laboratory test results depend largely on the quality of the specimen, the timing and the care with which it is collected, and the technical proficiency and experience of laboratory personnel.
Communication between Physician & Laboratory
Diagnostic microbiology encompasses the characterization of thousands of agents that cause or are associated with infectious diseases. The techniques used to characterize infectious agents vary greatly depending upon the clinical syndrome and the type of agent being considered, be it virus, bacterium, fungus, or other parasite. Because no single test will permit isolation or characterization of all potential pathogens, clinical information is much more important for diagnostic microbiology than it is for clinical chemistry or hematology. The clinician must make a tentative diagnosis rather than wait until laboratory results are available. When tests are requested, the physician should inform the laboratory staff of the tentative diagnosis (type of infection or infectious agent suspected). Proper labeling of specimens includes such clinical data as well as the patient's identifying data (at least two methods of definitive identification) and the requesting physician's name and pertinent contact information. Many pathogenic microorganisms grow slowly, and days or even weeks may elapse before they are isolated and identified. Treatment cannot be deferred until this process is complete. After obtaining the proper specimens and informing the laboratory of the tentative clinical diagnosis, the physician should begin treatment with drugs aimed at the organism thought to be responsible for the patient's illness. As the laboratory staff begins to obtain results, they inform the physician, who can then reevaluate the diagnosis and clinical course of the patient and perhaps make changes in the therapeutic program. This "feedback" information from the laboratory consists of preliminary reports of the results of individual steps in the isolation and identification of the causative agent.
Diagnosis of Bacterial & Fungal Infections
Specimens
Laboratory examination usually includes microscopic study of fresh unstained and stained materials and preparation of cultures with conditions suitable for growth of a wide variety of microorganisms, including the type of organism most likely to be causative based on clinical evidence. If a microorganism is isolated, complete identification may then be pursued. Isolated microorganisms may be tested for susceptibility to antimicrobial drugs. When significant pathogens are isolated before treatment, follow-up laboratory examinations during and after treatment may be appropriate.
A properly collected specimen is the single most important step in the diagnosis of an infection, because the results of diagnostic tests for infectious diseases depend upon the selection, timing, and method of collection of specimens. Bacteria and fungi grow and die, are susceptible to many chemicals, and can be found at different anatomic sites and in different body fluids and tissues during the course of infectious diseases. Because isolation of the agent is so important in the formulation of a diagnosis, the specimen must be obtained from the site most likely to yield the agent at that particular stage of illness and must be handled in such a way as to favor the agent's survival and growth.. Any type of microorganism cultured from blood, cerebrospinal fluid, joint fluid, or the pleural cavity is a significant diagnostic finding. Conversely, many parts of the body have a normal microbial flora that may be altered by endogenous or exogenous influences. Recovery of potential pathogens from the respiratory, gastrointestinal, or genitourinary tracts; from wounds; or from the skin must be considered in the context of the normal flora of each particular site. Microbiologic data must be correlated with clinical information in order to arrive at a meaningful interpretation of the results.
A few general rules apply to all specimens:
(1) The quantity of material must be adequate.
(2) The sample should be representative of the infectious process (eg, sputum, not saliva; pus from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not from its surface).
(3) Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions.
(4) The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful.
(5) Meaningful specimens to diagnose bacterial and fungal infections must be secured before antimicrobial drugs are administered. If antimicrobial drugs are given before specimens are taken for microbiologic study, drug therapy may have to be stopped and repeat specimens obtained several days later.
Microscopy & Stains
Microscopic examination of stained or unstained specimens is a relatively simple and inexpensive but much less sensitive method than culture for detection of small numbers of bacteria. A specimen must contain at least 105organisms per milliliter before it is likely that organisms will be seen on a smear. Liquid medium containing 105 organisms per milliliter does not appear turbid to the eye. Specimens containing 102-103 organisms per milliliter produce growth on solid media, and those containing ten or fewer bacteria per milliliter may produce growth in liquid media.
Gram staining is a very useful procedure in diagnostic microbiology. Most specimens submitted when bacterial infection is suspected should be smeared on glass slides, Gram-stained, and examined microscopically. On microscopic examination, the Gram reaction (purple-blue indicates gram-positive organisms; red, gram-negative) and morphology (shape: cocci, rods, fusiform, or other) of bacteria should be noted. The appearance of bacteria on Gram-stained smears does not permit identification of species. Reports of gram-positive cocci in chains are suggestive of, but not definitive for, streptococcal species; gram-positive cocci in clusters suggest a staphylococcal species. Gram-negative rods can be large, small, or even coccobacillary. Some nonviable gram-positive bacteria can stain gram-negatively. Typically, bacterial morphology has been defined using organisms grown on agar. However, bacteria in body fluids or tissue can have highly variable morphology.
Immunofluorescent antibody (IF) staining is useful in the identification of many microorganisms. Such procedures are more specific than other staining techniques but also more cumbersome to perform. The fluorescein-labeled antibodies in common use are made from antisera produced by injecting animals with whole organisms or complex antigen mixtures. The resultant polyclonal antibodies may react with multiple antigens on the organism that was injected and may also cross-react with antigens of other microorganisms or possibly with human cells in the specimen. Quality control is important to minimize nonspecific IF staining. Use of monoclonal antibodies may circumvent the problem of nonspecific staining. IF staining is most useful in confirming the presence of specific organisms such as Bordetella pertussis or Legionella pneumophila in colonies isolated on culture media. The use of direct IF staining on specimens from patients is more difficult and less specific.
Stains such as calcofluor white, methenamine silver, and occasionally periodic acid-Schiff (PAS) and others are used for tissues and other specimens in which fungi or other parasites are present. Such stains are not specific for given microorganisms, but they may define structure so that morphologic criteria can be used for identification. Specimens to be examined for fungi can be examined unstained after treatment with a solution of 10% potassium hydroxide, which breaks down the tissue surrounding the fungal mycelia to allow a better view of the hyphal forms. Phase contrast microscopy is sometimes useful in unstained specimens. Darkfield microscopy is used to detect Treponema pallidum in material from primary or secondary syphilitic lesions.
Culture Systems
For diagnostic bacteriology, it is necessary to use several types of media for routine culture, particularly when the possible organisms include aerobic, facultatively anaerobic, and obligately anaerobic bacteria. The standard medium for specimens is blood agar, usually made with 5% sheep blood. Most aerobic and facultatively anaerobic organisms will grow on blood agar. Chocolate agar, a medium containing heated blood with or without supplements, is a second necessary medium; some organisms that do not grow on blood agar, including pathogenic neisseria and haemophilus, will grow on chocolate agar. A selective medium for enteric gram-negative rods (either MacConkey agar or eosin-methylene blue [EMB] agar) is a third type of medium used routinely. Specimens to be cultured for obligate anaerobes must be plated on at least two additional types of media, including a highly supplemented agar such as brucella agar with hemin and vitamin K and a selective medium containing substances that inhibit the growth of enteric gram-negative rods and facultatively anaerobic or anaerobic gram-positive cocci.
Antigen Detection
Immunologic systems designed to detect antigens of microorganisms can be used in the diagnosis of specific infections. IF tests (direct and indirect fluorescent antibody tests) are one form of antigen detection .
Enzyme immunoassays (EIA), including enzyme-linked immunosorbent assays (ELISA) and agglutination tests are used to detect antigens of infectious agents present in clinical specimens. The principles of these tests are reviewed briefly here.
There are many variations of EIAs to detect antigens. One commonly used format is to bind a capture antibody, specific for the antigen in question, to the wells of plastic microdilution trays. The specimen containing the antigen is incubated in the wells followed by washing of the wells. A second antibody for the antigen, labeled with enzyme, is used to detect the antigen. Addition of the substrate for the enzyme allows detection of the bound antigen by colorimetric reaction. A significant modification of EIAs is the development of immunochromatographic membrane formats for antigen detection. In this format, a nitrocellulose membrane is used to absorb the antigen from a specimen. A colored reaction appears directly on the membrane with sequential addition of conjugate followed by substrate. In some formats, the antigen is captured by bound antibody directed against the antigen. These assays have the advantage of being rapid and also frequently include a built -in positive control. In latex agglutination tests, an antigen-specific antibody (either polyclonal or monoclonal) is fixed to latex beads. When the clinical specimen is added to a suspension of the latex beads, the antibodies bind to the antigens on the microorganism forming a lattice structure, and agglutination of the beads occurs. Coagglutination is similar to latex agglutination except that staphylococci rich in protein A are used instead of latex particles; coagglutination is less useful for antigen detection compared with latex agglutination but is helpful when applied to identification of bacteria in cultures.Latex agglutination tests are primarily directed at the detection of carbohydrate antigens of encapsulated microorganisms. Another form of EIA, to detect antibody, is immunoblotting ("Western blot"), whereby defined antigens are placed on strips of nitrocellulose paper. Following incubation with the test antibody-containing specimen, the strip is further treated with an enzyme-labeled antibody, usually from another animal, against the test antibody. Addition of the substrate for the enzyme allows detection of the antigen-specific bound antibody by colorimetric reaction. Western blot tests are used as the specific tests for antibodies in HIV infection and Lyme disease.
Molecular Diagnostics
The principle behind early molecular assays is the hybridization of a characterized nucleic acid probe to a specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. For example, single-stranded probe DNA (or RNA) is used to detect complementary RNA or denatured DNA in a test specimen. The nucleic acid probe typically is labeled with enzymes, antigenic substrates, chemiluminescent molecules, or radioisotopes to facilitate detection of the hybridization product. By carefully selecting the probe or making a specific oligonucleotide and performing the hybridization under conditions of high stringency, detection of the nucleic acid in the test specimen can be extremely specific. Such assays are currently used primarily for rapid confirmation of a pathogen once growth is detected, eg, the identification of Mycobacterium tuberculosis in culture using the Gen-Probe Inc. (San Diego, CA) DNA-probe.
Identifying Bacteria Using 16S rRNA
The 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in each organism. Labeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double-stranded hybrid is measured. This technique is widely used for the rapid identification of many organisms. Examples include the most common and important Mycobacterium species, Coccidioides immitis, Histoplasma capsulatum, and others. Molecular diagnostic assays that use amplification techniques have become widely used and are evolving rapidly.
Target Amplification Systems
In these assays, the target DNA or RNA is amplified many times. The polymerase chain reaction (PCR) is used to amplify extremely small amounts of specific DNA present in a clinical specimen, making it possible to detect what were initially minute amounts of the DNA. PCR uses a thermostable DNA polymerase to produce a twofold amplification of target DNA with each temperature cycle. The DNA extracted from the clinical specimen along with sequence-specific oligonucleotide primers, nucleotides, thermostable DNA polymerase, and buffer are heated to 90–95 °C to denature (separate) the two strands of the target DNA. The temperature in the reaction is lowered, usually to 45–60 °C depending upon the primers, to allow annealing of the primers to the target DNA. Each primer is then extended by the thermostable DNA polymerase by adding nucleotides complementary to the target DNA yielding the twofold amplification. The cycle is then repeated 30–40 times to yield amplification of the target DNA segment by as much as 105 to 106fold. The amplified segment often can be seen in an electrophoretic gel or detected by Southern blot analysis using labeled DNA probes specific for the segment or by a variety of proprietary commercial techniques.
PCR can also be performed on RNA targets, which is called reverse transcriptase PCR.
The enzyme reverse transcriptase is used to transcribe the RNA into complementary DNA for amplification.
PCR assays are available commercially for identification of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, cytomegalovirus, enteroviruses, and many others. An assay is available for HIV-1 viral load testing also. There are many other "in-house" PCRs that have been developed by individual laboratories to diagnose infections. Such assays are the tests of choice to diagnose many infections—especially when traditional culture and antigen detection techniques do not work well. Examples include testing of cerebrospinal fluid for herpes simplex virus to diagnose herpes encephalitis and testing of nasopharyngeal wash fluid to diagnose Bordetella pertussis infection (whooping cough).
A major consideration for laboratories that perform PCR assays is to prevent contamination of reagents or specimens with target DNA from the environment, which can obscure the distinction between truly positive results and falsely positive ones because of the contamination.
|
|
مخاطر خفية لمكون شائع في مشروبات الطاقة والمكملات الغذائية
|
|
|
|
|
"آبل" تشغّل نظامها الجديد للذكاء الاصطناعي على أجهزتها
|
|
|
|
|
تستخدم لأول مرة... مستشفى الإمام زين العابدين (ع) التابع للعتبة الحسينية يعتمد تقنيات حديثة في تثبيت الكسور المعقدة
|
|
|