A Wetpaint Site | TV Fandex 100

Diagnosing Infections

Chapter Overview

The ability to identify microbes that are responsible for a patients symptoms is central to infectious disease microbiology. Diagnosis might be considered an art and a science, involving multiple health care providers and clinical personnel.

In order to successfully identify an infectious agent, the specimen containing that agent must be properly collected, handled, and stored.
1. Accurate diagnosis depends on the ability to differentiate the causative organism from normal flora or contaminating microbes.
Most diagnostic tests fall into two categories: presumptive data, which place the microbe in a preliminary category, and more specific data which provide definitive evidence of a species.
1. G+C relative to A+T can place a microbe in a preliminary category, but specific DNA sequences could identify it to a species level.
Microbiologists use phenotypic, immunological and genotypic techniques to identify bacteria.

Phenotypic identification methods assess a microbe's appearance, growth characteristics, and/or chemicals and enzymes.
Most lab tests are phenotypic identification methods. Usually several tests need to be done in a sequence before identification can be made. A "dichotomous key" of "flowchart" guides the testing.
Specimens are often inoculated into differential and selective media to define characteristics such as fermentation patterns and reactions (lysis) in blood.
Microscopic observation of specimens is a rapid method to determine preliminary characteristics of a microbe.

DNA sequences that are unique to an organism can be used to “probe” for that organism.
1. PCR is often used to amplify DNA present in samples.
2. PCR depends on DNA polymerase from hot springs bacteria Thermophilus aquaticus (Taq polymerase)
3. Here are two videos: one with a good explanation of how PCR works, and the second is a corny song of scientists singing about how great PCR is ("PCR, when you need to find out who your daddy is...").
Serology deals with in-vitro diagnostic testing of serum, has a high degree of specificity and sensitivity, and is based on the specificity an antibody has for its antigen.
1. Agglutination testing is based on the cross-linking of antigens and antibodies to form visible clumps
2. Precipitation reactions involve the precipitation of a soluble antigen by an antibody.
3. Western blot analysis uses antibodies to identify specific proteins that have been transferred onto a filter.
4. Complement fixation tests can be used to determine the presence of specific antigen-antibody complexes.
5. Monoclonal antibodies can be labeled with a fluorescent dye and used to detect a specific antigen in a solution or used to react with an Fc portion of another antigen.
6. The ELISA involves the interaction of antibodies and antigens and can be used to determine an unknown antibody (indirect) or antigen (direct or capture).

Tests by Type

Microbiologists use three categories of techniques to diagnose infections: phenotypic, genotypic, and immunological.

Phenotypic Tests	Immunological Tests	Genotypic Tests
Direct examination of specimen -Gram stain -Acid-fsat stain -DFA (Direct Fluorescence Antibody) -Direct antigen testing (rapid testing)	Agglutination Testing -Widal test -Latex agglutination	Nucleic acid sequencing and rRNA analysis
Biochemical testing -Carbohydrate fermentation -Hydrolysis of gelatin, starch -Enzyme actions (catalase, oxidase, coagulase) -Byproducts of metabolism	Precipitation testing	Polymerase chain reaction
Phage typing	Western Blot	G+C base composition
Animal inoculation	Compliment fixation
Antimicrobial sensitivity	Toxin neutralization -Treponema pallidum immobilization (TPI) -Quellung
	Fluorescent antibodies/immunofluorescent testing -Direct vs. indirect
	Immunoassays -Radioimmunoassay (RIA) -Enzyme-Linked Immunosorbent Assay (ELISA)
	T cell and B cell differentiation testing
	In vivo testing -Tuberculin test

Phenotypic Methods
The main phenotypic methods include the direct examination of specimens, observing the growth of specimen cultures on special media, and biochemical testing of specimen cultures.
Microscopic Morphyology-Traits that can be valuable aids to identification of cell shape and size, Gram-stain reaction, acid-fast reaction and special structures, including endospores, granules, and capsules. Electron microscopes can pinpoint additional features such as: cell wall flagella, pili, and fimbriae.

Macroscopic Morphology-Traits that can be assessed with the naked eye are also useful in diagnosis. These include the appearance of colonies, including texture, shape, size, pigment, speed of growth, and patterns of growth in broth and gelatin.

Physiological/Biochemical Characteristics-Bacterial identification: Enzymes and other biochemical properties of bacteria are reliable and stable expressions of the chemical identity of each species.Diagnostic tests exist for determining the presences of specific enzymes and to assess nutritional and metabolic activities. Test examples: fermentation of sugars, capacity to digest or metabolize complex polymers such as proteins and ploysaccharides; production of gas; presence of enzymes such as catalase, oxidase, and decarboxylase; and sensitivity to antimicrobic drugs.

Chemical Analysis-This involves analyzing the types of specific structural substances that the microorganism contains, such as the chemical composition of peptides in the cell wall and lipids in the membrane.

Genotypic Methods-Identifying and classifying bacteria. There are many advantages of genotypic methods over phenotypic methods, when they are available. The primary advantage is that actually culturing the microorganisms is not always necessary. There are many microorganisms we can't grow in the lab, compared to how many we can. Another advantage is that genotypic methods are increasingly automated, and results are obtained very quickly, often with more precision than with phenotypic methods.

Immunological Methods-bacteria and other microbes have surface and other molecules called antigens that are recognized by the immune system.On immune response to antigens is the production of molecule called antibodies that are designed to bind tightly to the antigens.The nature of antibody response can be determined from blood or other tissue samples. The presence of specific antibodies to a suspected pathogen is highly suggestive of infection.This is an easy test for microbes, especially in the case of viral infections. (Example: HIV testing). Laboratory kits immediately identify the number of pathogens.

Specimen Collection
In sites that normally contain resident microflora, care should be taken to sample only the infected site and not the surrounding areas. For example, throat and nasopharyngeal swabs should not touch the tongue, cheek or saliva. Saliva is an especially undesirable contaminant because it contains millions of bacteria, of which are normal flora. Sputum, the mucous secretion that coats the lower respiratory surfaces, especially the lungs, is discharged by coughing or taken by a catheterization to avoid contamination with saliva.
Also the mucous lining of the vagina, cervix, or urethra can be sampled with a swabbed or applicator stick.
Additional sources of specimens are the vagina, eye, ear canal,nasal cavity (all by swab), and diseased tissue that has been surgically removed (biopsied).

Urine is taken aseptically from the bladder with a catheter. Another method is called a "clean catch" is taken by washing the external urethra and collecting the urine in midstream. Some diagnostic techniques require first-voided "dirty catch" urine.

Sterile materials such as blood. cerebrosspinal fluid, and tissue fluid must be taken by sterile needle aspiration.

Overview of Laboratory Techniques
1.Direct testing using a microscope, immunological, or genetic methods that provide immediate clues as to the identity of the microbe or microbes in sample and 2. cultivation, isolation, and identification of pathogens using a wide variety of general and specific tests (such as blood or other fluids).

Most tests fall into two categories:presumptive data, which place the isolated microbe in a preliminary category such as genus, and more specific, confirmatory data, which provide more definitive evidence of a species.

Some diseases are diagnosed with out the need to identify microbes from specimens. Serological tests on a patients serum can detect signs of an antibody response. One method that clarifies whether a positive test indicates current or prior infection is to take two samples several days apart and see if the antibody titer is raising. Skin testing can pinpoint a delayed allergic reaction to a microorganism. These tests are important in screening the general population for exposure to an infectious agent such as rubella or tuberculosis.

Immediate Direct Examination of Specimens
Direct microscope observation of a fresh or stained specimen is one of the most rapid methods of determining characteristics. Stains most often employed for bacteria are the gram stain, they do not work on some organisms though. Direct florescence antibody (DFA) test can highlight the presence of the microbe in patient specimens by means of labeled antibodies. This test is useful for bacteria such as syphilis spirochete, that are not readily cultivated in a laboratory or if rapid diagnosis is essential for the survival of a patient.

Isolation Media
In most cases, specimens are also inoculated into differential media that define such characteristics as reactions in blood(blood agar) and fermentation patters (mannitol salt and MacConkey agar. A patients blood is usually cultured in a special bottle of broth that can be periodically sampled for growth. Work must be done from isolated colonies or pure cultures, working with mixed or contaminated cultures give misleading and inaccurate results.From such isolates, clinical microbiologist obtain information about a pathogen's microscopic morphology and staining reactions, culture appearance, motility, oxygen requirements, and biochemical characteristics.

General Guidelines	For Specimen	Collection
Type of Specimen	Collection Method	Comments
Abcess of skin or membrane or decubitus ulcer	Debride surface with sterile wipe; suction fluid with sterile needle and syringe	Aspiration of tissue samples preferred to swab; transfer to anaerobic transport system.
Anaerobic cultures	Collect from deeper tissue below the surface, using sterile syringe	Sample must not be exposed to air; transported immediately in anaerobic system to lab
Blood	prep area of skin eith iodine; use vacutainer blood collection tubes	Inoculate blood culture bottle immediately
Bone marrow	Prepare site for surgical incisiton; extract sample with special needle.	Most samples are taken from sternum or ilium; placed in blood bottle for culture
Cerebrospinal fluid (CSF)	Remove CSF aseptically by lumbar puncture	Place CSF into sterile tubes; do not refigerate; transport immediatiely to lab for processing
Feces	Take a small specimen into sterile container, cover, and transport	Refrigerate samples held over one hour; culture is mainly to rule out enteric pathogens; special kits available to detect systs and trophozoites of protozoans and ova and larvae of intestinal worms
Genital/urinary tract	Sterile swab of cervical mucus; swab urethral membrane or insert into lumen and twist.	Plate directly onto selective culture media with high CO2 atmosphere; or collect wiht anaerobic transport swab system; process immediately.
Respiratory tract, lower	Have patient cough to loosen phlegm and expectorate sputum into sterile cup for transport	Patient should brush teeth and rinse mouth with water prior to any sampling. If coughing does not work, induce sputumby having patient inhale sterile saline from a nebulizer.
Urine	Sampling is usually done with sterile catheter or by clean midstream cath into a sterile container.	Patient must wash hands and clean external genitalia, separate labia (female) or pull back foreskin (male) and urinate for a short time; followed by collection of 100-200 cc sample (midstream); samples not immediately processed must be refigerated.
	Catheter must be sterile and inserted into bladder; urine is drainded into sterile container	Clean and rinse the urethral opening; only a specimen catheter should be used ot sample for bladder infection; patients wiht indwelling catheters will always have microbes in their bladders.

Questions: Q&A~~Home Page~~Top of Page

1. Which of the following methods is most sensitive for identifying different strains of a microbe?
a. microscopic examination
b. radioimmunoassay
c. DNA typing
d. agglutination test

2. In agglutination reactions, the antigen is a ______; in precipitation reactions, it is a ________.
a. soluble molecule, whole cell
b. whole cell, soluble molecule
c. bacterium, virus
d. protein, carbohydrate

3. Which reaction requires complement?
a. hemagglutination
b. precipitation
c. hemolysis
d. toxin neutralization

4. A patient with a _______ titer of antibodies to an infectious agent generally has greater protection than a patient with a ________ titer.
a. high, low
b. low, high
c. negative, positive
d. old, new

5. Direct immunofluorescence tests use a labeled antibody to identify _____.
a. an unknown microbe
b. an unknown antibody
c. fixed complement
d. agglutinated antigens

6. The Western blot test can be used to identify
a. unknown antibodies
b. unknown antigens
c. specific DNA
d. both a and b

7. An example of an in vivo serological test is
a. indirect immunofluorescence
b. radioimmunoassay
c. tuberculin test
d. complement fixation

Definitions

Phenotypic: The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences.
Genotypic: the genetic constitution of an individual, that is the specific allele makeup of the individual, usually with reference to a specific character under consideration
Probes: Mechanical devices equipped with sensors that enable us to gather information without direct human contact.
Serology: The branch of science dealing with the measurement and characterization of antibodies and other immunological substances in body fluids, particularly serum.
Widal Test: the Widal test is a presumptive serological test for Enteric fever or Undulant fever.
Titer: A measurement of the amount of antibodies in the blood
Lysin: any substance (such as an antibody) or agent that can cause lysis
Complement Fixation: an immune response in which an antigen-antibody combination inactivates a complement
Treponema pallidum immobilization (TPI): A test for syphilis in which an antibody other than Wassermann antibody is present in the serum of a syphilitic patient; in the presence of complement, the patients serum causes the immobilization of actively motile Treponema pallidum obtained from testes of a rabbit infected with syphilis. Also called TPI test.
Immunoassays: Are extremely sensitive alternative methods that permit rapid and accurate measurements of trace antigen or antibody.

flattail	Latest page update: made by flattail , Jun 23 2009, 5:19 PM EDT (about this update About This Update Moved from: Chapter Notes - flattail No content added or deleted. - complete history)
	Keyword tags: analysis diagnosis Diagnostic test G+c Genotype identification methods PCR phenotype TPI traits Treponema western blot Widal Test More Info: links to this page

Threads for this page

Post a new thread

There are no threads for this page. Be the first to start a new thread.