Lab 8

-all the broths, all the tubes, all the carbohydrates, all the indicators, all the reagents, all the selective and differential media-

• Ready to use
• Microtube system containing dehydrated media
  • Rehydration occurs upon addition of a measured bacterial suspension
  • Bacteria react with the contents of each of the microtubes
  • Various indicators within the microtubes allow for interpretation of results within 18-24 hours of inoculation, when incubation occurs at 35-37^oC.
• 21 standard biochemical tests performed
• Uses one single colony of bacteria from plating medium
• Standardizes testing methods and interpretation of test results
  • The reactions are converted into a seven digit code
  • The code can then be fed into the manufacturer's database (via a touchtone phone) and the computerized voice provides the identification, usually down to the genus and species
  • The code can also be looked up in a reference book
• Allows for valid comparisons of results both within a lab and across labs throughout the world

Thus, the long-accepted, time-consuming system of identifying bacteria has undergone some changes. It is now possible to identify certain bacteria (not all) based on serodiagnostic technology. Serodiagnostic technology involves antigen/antibody reactions. Recall from Lab 6 the "Overview of the Diagnosis of Bacterial Disease:"

 An Overview of the Diagnosis of Bacterial Disease

Today we will cover a variety of serodiagnostic tests:

• Staphaurex
• ELISA
• Western Blot
• Immunofluorescence

Staphaurex

This test is a "rapid slide agglutination" test that can distinguish Staphylococcus aureus from other types of Staphylococcus species.

The Staphaurex test kit is made by Murex, a company specializing in these kinds of immunological tests.

S. aureus bacteria have two kinds of proteins on their cell walls (that are of use in this test)

• Clumping Factor (coagulase factor)
• Protein A

• Fibrinogen (a clotting protein)
• Antibody to Protein A (called Rabbit IgG)

When the test result looks like the illustration above left, the test is considered to be positive for S. aureus. When the test result looks like the illustration above right, the test is considered to be negative for S. aureus. Controls are always run.

Below is a pictorial sequence of the steps in this test:

Note that this is the third definitive (diagnostic) test method you have learned for the presumptive identification of Staphylococcus aureus. The three definitive tests are:

• Fermentation of mannitol on MSA (agar turns yellow)
• Coagulase positive
• Staphaurex positive

There are two forms of this test:

• Direct ELISA is a test that is measuring the presence of the antigen in the patient
• Indirect ELISA is a test that is measuring the presence of the antibody in the patient

This test, sometimes called an enzyme immunoassay (EIA) procedure, is an interesting application of the antigen/antibody interaction methodology. ELISA stands for "enzyme-linked immunosorbent assay." To illustrate its principle, we shall look specifically at one use of this test, the initial screen for human immunodeficiency virus (HIV) antibody detection in patients' plasma or serum. Based on what you just learned, do you expect this type of test to be a direct or indirect ELISA test (what is the test measuring?).

Recall that human antibodies are found in the liquid portion of blood. This component is called plasma or serum, depending on the presence or absence of clotting factors. Since clotting factors are not essential to the success of this test, either plasma or serum may be used.

Below is a photograph of the ELISA testing plate. This plate measures 8 cm by 12 cm and contains eight rows of 12 test wells, for a total of 96 wells. The wells are about one centimeter deep, deep enough for a few drops of serum and test reagents.

To perform the test:

1. Inactivated HIV antigens are pre-coated at the manufacturer's onto each well's bottom on the ELISA plate (that's 96 inactive viruses glued to the bottom of the wells!).
2. Add a drop of patient serum (note that the plate allows for 94 patients to be tested at one time PLUS a positive and negative control).
   • If the patient is infected with HIV, his HIV antibodies will attach to the HIV antigen at the bottom of the well.
   • If the patient is not infected with HIV, there will not be any attachment of antibody to the antigen at the bottom of the well.
3. Wash the well.
   • Anything not attached to the inactivated antigen (virus) is washed into the sink.
   • If the patient is infected with HIV, his HIV antibodies remain stuck to the HIV antigen at the bottom of the well.
   • If the patient is not infected with HIV, there is nothing left in the well!
4. Add the first of two ELISA reagents..
   • This first reagent is an antibody to the HIV antibody. It is called an antihuman immune serum globulin.
   • Cool, huh?
   • It is attached to an enzyme.
   • If the antibody to the HIV antibody comes in contact with the patient's antibody that is stuck to the inactivated antigen (virus), it becomes stuck as well!
   • If the antibody to the HIV antibody does not come in contact with the patient's antibody (as in the case of a negative HIV patient), then nothing happens here.
5. Wash the well.
   • Anything not attached to the antihuman immune serum globulin that is attached to the HIV antibody that is stuck to the inactivated antigen (virus) is washed into the sink.
   • If the patient is infected with HIV, the antihuman immune serum globulin has a death grip on the patient's HIV antibodies that are stuck to the HIV antigen at the bottom of the well, and this complex remains in the well. (Can you get a visual on this?!)
   • If the patient is not infected with HIV, there was nothing for the antihuman immune serum globulin to stick onto, and the well is empty.
6. Add the second ELISA reagent.
   • This second reagent contains a substrate for the above enzyme and a chromogen (guess what chromogens give rise to-color!). If the enzyme and substrate combine, a color change will occur. This color change is visible to the naked eye.
   • If the patient is infected with HIV, the substrate comes in contact with the enzyme that is linked to the antihuman immune serum globulin, and a chemical reaction occurs that causes a color change (thank you, chromogen!).
   • If the patient is not infected with HIV, there is no color change, because the enzyme was washed away with the last washing.

In essence, this is a sandwich test:

• You've got HIV antigen (virus) on one end.
• You've got antibody to HIV antibody on the other end.
• In the middle is the "meat," the HIV antibody, which is what you are looking for!

Questions to think about:

• What if the patient is only recently infected with HIV and has not had time to make antibodies to HIV?
• What if the patient is in the end-stages of acquired immune deficiency syndrome (AIDS) and can no longer make antibodies to HIV?
• What if the patient has been exposed to an antigen that happens to cause an antibody very similar to the antibody against HIV?

Note that this test is not definitive for HIV. This test is a screening test. When positive, it is always followed by a confirmatory test. In most cases, the confirmatory test is a Western Blot test.

This test can also be a direct test (looking for the antigen) or an indirect test (looking for the antibody). Again, we will use HIV antibody testing as our example.

The differences between the antibodies measured by ELISA and the antibodies measured here are:

• ELISA tests look for antibody to the whole virus
• Western Blot tests look for antibodies directed against specific viral proteins
  • These viral proteins make up the human immunodeficiency virus
  • There are six of them, rather than just one general antigen
    • gp 160
    • gp 120
    • gp 55
    • gp 41
    • p 55
    • p 31
  • They are very specific to the virus
  • Any antibodies detected are specific to these six antigens

In this test, the manufacturer takes HIV positive lymphocytes and lyses them. 'Lyses' means that the cells are caused to rupture, thus releasing their contents. The ruptured contents are then applied onto a special agar medium.

An electrical current is run through this agar medium for a pre-determined period of time.

• The current causes the ruptured cells' material to move through the agar, according to the material particles' size and electrical charge.
• Thus, when the electrical current is stopped, each particle will have come to a halt, based on these two characteristics.
• The largest particles move the slowest and will be found closest to the application point.
• The smallest particles move the fastest and will be the furthest away from the application point.
• The more electronegative particles will move faster than the less electronegative particles.
• This process is called "electrophoresis."

Once the electrophoresis of the ruptured cell's particles has taken place, a "purification" step is required. The electrophoresed agar, containing the particles staggered according to size and charge, is "blotted" onto another membrane (still containing the same sequence of particles). This new membrane is:

• "Blocked" at all sites of the membrane that do not actually contain the electrophoresed particles.
• The blocking prevents any antibodies from the patient from sticking.
• Only the electrophoresed particles are left unblocked.
• Thus, if a specific antibody to an electrophoresed particle is present in the patients' plasma (or sera), that antibody will be able to stick to the particle.

Now, just like with the ELISA test, a washing step occurs. This removes all traces of the patient's serum from the membrane, EXCEPT for any antibodies that attached to the electrophoresed particles on the membrane.

If you have been following this closely, you know the rest of the story. In order to detect any patient antibodies that may be attached to the HIV protein particles on the membrane, a second antibody, the antihuman immune serum globulin, is now added onto the membrane. This second antibody:

• Is called a "conjugate"
• Is composed of an antibody to the antibody (that is attached to the electrophoresed particle) and an enzyme
• The conjugate attaches to any antibody that is sticking to the membrane
• If there is no antibody, nothing attaches

There is a second wash step. This step removes any unattached conjugate. Following the second washing, a substrate is added, which causes a precipitated residue to form that can be visualized as a band, if it comes in contact with the enzyme (which is part of the conjugate). If there is no enzyme present, there is no precipitated band formed.

Now, let's see how closely you have been following the above explanation by having you study a real life example below. The six blotted proteins are found in all six of the columns below (each column represents one membrane containing the blotted, unblocked proteins). Lane 1 is the positive control. Lane 2 is the negative control. Lanes A, B, and C contain unknowns (three patients). Can you tell whether any of the patients is positive for antibodies against any of these six blotted proteins? Remember, a band is a sign of the presence of patient antibody to particle antigen.

• Lane 1, HIV+ serum (positive control)
• Lane 2, HIV- serum (negative control)
• Lane A, Patient A
• Lane B, Patient B
• Lane C, Patient C

To help with the interpretation of test results, the Centers for Disease Control and Prevention (CDC) defined the criteria for HIV positivity. These standards are used by all HIV test sites, thus guaranteeing consistency in test results, regardless of test locale.

The criteria are:

• Bands that are found at either p31 or p24 AND at either gp160 or gp120 are indicative of a positive Western Blot Assay for HIV antibody
• No bands at all are indicative of a negative Western Blot Assay for HIV antibody
• The presence of bands in any pattern different from above is indicative of an indeterminate Western Blot Assay for HIV antibody (the recommendation is for a repeat test at a later date)

Now go back and see if you can determine if any of the above unknowns has a positive Western Blot Assay, according to the above criteria!

Immunofluorescence

This is yet another technique for "labeling" the antigen/antibody complex such that the investigator can visualize it. Up until now, color changes (ELISA) and precipitation of sediment (Western Blot) have been used to allow for visualization of the immune complex. Immunofluorescence is the name given to attaching a fluorescent dye (immunofluorescent reagent) to the reagent antibody. The investigator then looks for the presence of fluorescence (using a fluorescent microscope and ultraviolet light) as an indicator of the reaction.

Just as before, there are two types of immunofluorescence techniques. In direct immunofluorescence, the investigator is looking for the antigen itself (either bacterial or viral) in the patient. Thus, the reagent consists of an antibody to that antigen.

In indirect immunofluorescence, the investigator is looking for the presence of an immune response. That is, does the patient have antibody to the bacteria or virus present? In this instance, an antibody to the antibody will be used. Remember, this kind of antibody is called an antihuman immune serum globulin, and it is attached to the fluorescent dye.

Summary:

Direct testing:

• Looking for antigen (bacterial or viral).
• Add antibody to patient sample, attached to some kind of labeling molecule that allows for visualization.

• Looking for antibody (to the bacteria or virus).
• Antigen is present in the test kit.
• Add patient serum.
• Add antihuman immune serum globulin, attached to some kind of labeling molecule that allows for visualization.

Antimicrobial Susceptibility Testing

When a microorganism has been isolated from a patient specimen, the obvious question is 'how can we eliminate this organism'? The goal of antimicrobial susceptibility testing is to predict the success or failure of antibiotic therapy in the patient.

'In the patient' is considered an in vivo (living) situation. Unfortunately, all tests are performed in vitro (literally 'in glass tubes,' it means outside of the patient). Consequently, results should be viewed in the context of the patient's clinical information and prior clinician experience with the selected antimicrobial.

All antimicrobial susceptibility testing is performed under standardized test conditions. The results must be reproducible. This means that, regardless of who performs the test, which lab the test is performed in, the circumstances surrounding the test performance, or any other variable, the test results will always be the same for that particular sample.

Today we will look at one type of antimicrobial susceptibility testing, the Kirby-Bauer disc diffusion test.

Kirby-Bauer Test

A large agar plate is inoculated uniformly with a standardized amount of bacteria. 'Uniformly' means that there is an equal amount of bacteria throughout the entire plate. Some people call this a "lawn." 'Standardized' means that there are policies and procedures in place that have been accepted by microbiologists throughout the world as to the exact quantity of bacteria that is to be used for the inoculum. This quantity is consistently adhered to.

Following this, small filter paper disks (about 6 mm in diameter), each of which has been impregnated with a standard amount of antibiotic, are placed onto the agar plate. The plate is incubated. During incubation, the chemotherapeutic agents diffuse from the disks into the agar. The concentration of the agent is greatest, of course, closest to the disk and least at the furthest distance from the disk.

Once incubation is completed, microbiologists measure zones of inhibition, in mm, as an indicator of the effectiveness of the agent. A zone of inhibition is a circular area radiating from the disk in which growth of the microorganism has been completely inhibited. The diameter of this zone is measured and then compared to a standard table in order to correctly interpret the results.

Antimicrobial susceptibility of the microorganism is defined as the organism either being sensitive, intermediate, or resistant to the chemotherapeutic agent.

• Sensitive means that the organism is susceptible to the antibiotic. The implication to the clinician here is that an infection caused by the strain of microorganism isolated by the laboratory may be treated with the usual dosage of the tested antimicrobial agent.
• Resistant means that the antibiotic does not inhibit the organism. The implication to the clinician here is than an infection caused by the strain of microorganism isolated by the laboratory will not respond appropriately to the normal dosage schedule for this particular antimicrobial agent.
• Intermediate defines the continuum of susceptibility and resistance. Antimicrobial susceptibility is not black and white-sensitive or resistant. There is a gray area that is best interpreted by the clinician, based on his experience with the drug and his patient's clinical picture. Infections caused by organisms in this category may or may not respond to therapy with the tested agent.

Take Out Food for the Brain:

1 Figures are Joint United Nations HIV/AIDS Programme estimates as of December 1999. Note that the estimated number of people living with AIDS reported in the table for the world includes both people with AIDS and those who are HIV infected, but who may not have developed the clinical manifestations defining AIDS.

2 Figures reported by the U.S. Centers for Disease Control and Prevention (CDC) as of June 30, 1999 except for estimate of HIV infection to date, which was reported in the Journal of the American Medical Association (v. 276) as of 1992, and the estimate of new infections per year, which was reported by the CDC in 1997.

3 Figures are as of Sept. 30, 1999 reported by the State Department of Health Services, Office of AIDS.

4 Figures are as of Sept. 30, 1999 reported by the San Francisco Department of Public Health except for estimate of HIV infections to date and estimated infections per year, which were reported in a 1997 HIV Consensus Report, also issued by the Department of Public Health.

Location	MSM7	IDU8	MSM/ IDUs	Hetero- sexual*	Trans- fusions	Hemo- philiacs	Pediatric ( 0-12)	Other/ Unknown
United States	334,073	179,228	45,266	70,582	8,430	5,010	8,596	60,159
California	80,377	11,471	10,166	4,960	1,582	528	581	4,420
San Francisco	20,647	1,774	3,178	326	202	43	51	177

(7 Men who Have Sex with Men)
(8 Injection Drug Use)
(*Heterosexual transmission is on the rise; the number of new infections among women is now almost equal to the number of new infections among men)