CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Serology Manual
Schedule
Module 1: Mechanisms of Immunity
Objectives
Study Questions
Module 2: Serological Test Methods
Objectives
Study Questions
Module 3: Infectious Diseases
Spirochetes
Objectives
Study Questions
Streptococcal
Objectives
Streptococcal Antibody Testing Handout
Study Questions
Viral and Parasitic
Objectives
Immunological Testing for Viral Infections Handout
Immunological Testing for Parasitic Infections
Study Questions
HIV
Objectives
Human Immunodeficiency Virus handout
Study Questions
Module 4: Autoimmunity and Autoimmune Diseases
Objectives
Autoimmunity & Autoimmune Disorders handout
Study Questions
CLS 419 Clinical Microbiology II
Serology
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CLS 419 Clinical Microbiology II – Serology Section
University of Nebraska Medical Center
Clinical Laboratory Science Program
Schedule for 2007-2008
Textbook: “Clinical Immunology and Serology – A laboratory perspective”, Stevens, 2nd edition.
To be completed during Rotation II Serology Week:
(The serology grade is included in CLS 419 Clinical Microbiology II)
Complete the assigned readings (textbook, handouts, and websites) and study questions (yellow sheets)
for each of the following modules:
1. Mechanisms of Immunity
a. Read: Stevens, Chapters 1-7, 13
b. Review student laboratory materials from Immunology
c. Do Study Questions (yellow sheets in manual, key on Blackboard)
d. Do Study Questions in Stevens 2nd edition textbook:
Chapter 1, #5-7, page 8
Chapter 2, #1-13, page 22
Chapter 3, #1, #3-12, page 43
Chapter 4, #1-8, #10, page 57
Chapter 5, #1-15, page 75
Chapter 6, #11 page 90;and
Chapter 7, #1-4, #6-12, #14, pages 111-112
2. Serological Test Methods
a. Read: Stevens, Chapters 8-11
b. Review student laboratory materials from Immunology
c. View “Laboratory Methods Tutor” (Univ. of Washington MTS LTL Site)
d. Do Study Questions (yellow sheets in manual, key on Blackboard)
e. Do Study Questions in textbook:
Chapter 8, #11-14, page 126
Chapter 9, #1-4, 6-7, 9-13, page 140
Chapter 10, #1-3, 5-7, 9-11, page 154
Chapter 11, #1-3, 5, 7-12, page 171
3. Infectious Diseases: Spirochetes
a. Read: Stevens, Chapters 19, and Mahon, Chapter 20
b. View Spirochetes PowerPoint on Blackboard
c. View the following CDC webpages:
Syphilis Fact Sheet (http://www.cdc.gov/std/syphilis/STDFact-Syphilis.htm)
Lyme Disease (http://www.cdc.gov/ncidod/dvbid/lyme/index.htm)
Relapsing Fever (http://www.cdc.gov/ncidod/dvbid/RelapsingFever/index.htm)
Leptospirosis (http://www.cdc.gov/ncidod/dbmd/diseaseinfo/leptospirosis_g.htm)
d. Do Study Questions (yellow sheets in manual, key on Blackboard)
e. Do Study Questions in textbook:
Chapter 19 #1-17, pages 310-311
4. Infectious Diseases: Streptococcal
a. Read: Stevens, Chapters 20
b. Read Streptococcal Antibody Testing Handout
c. Do Study Questions (yellow sheets in manual, key on Blackboard)
e. Do Study Questions in textbook:
Chapter 20, #1, 4-8, page 322
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Schedule
Page 2
5. Infectious Diseases: Viral
a. Read: Stevens:
Chapter 21 - Rubella, CMV, Herpes Simplex I & II, TORCH, EBV (IM), Hepatitis
Chapter 22 - HIV
b. Review student laboratory material:
EBV (IM): hematology notes
c. Read: Immunologic Testing for Viral Infections Handout and
HIV Handout
d. View the following CDC webpages:
West Nile Virus (http://www.cdc.gov/ncidod/dvbid/westnile/)
HIV (http://www.cdc.gov/hiv/)
e. Do Study Questions (yellow sheets in manual, key on Blackboard)
f. Do Study Questions in textbook:
Chapter 21, #5-9, page 343
Chapter 22 #1-15 pages 367-368
7. Infectious Diseases: Parasitic
a. Read: Stevens, Chapter 23, pgs. 372-374
b. Read Immunologic Testing for Parasitic Infections Handout
c. View CDC webpage:
http://www.cdc.gov/ncidod/dpd/parasites/toxoplasmosis/factsht_toxoplasmosis.htm
d. Do Study Questions (yellow sheets in manual, key on Blackboard)
e. Do Study Questions in textbook:
Chapter 23, #3-6, page 384
8. Autoimmunity and Autoimmune Diseases
a. Read: Stevens, Chapter 14, pgs. 212-228
b. Read Autoimmunity and Autoimmune Disorders Handout
c. View: Immunology: Antinuclear Antibodies & Mouse Stomach-Kidney (Univ. of Washington
Website http://www.medtraining.org/ltl)
d. Do Study Questions (yellow sheets in manual, key on Blackboard)
e. Do Study Questions in textbook
Chapter 14, #1-12, page 232
The examination at the end of this week will focus on the following objectives:
1. Mechanisms of Immunity
2. Immunologic Methods
3. Infectious Disease: Spirochetes
4. Infectious Disease: Streptococcal Antibody Testing
5. Infectious Disease: Miscellaneous Serology Testing (including TORCH)
6. Infectious Disease: Epstein-Barr Virus (EBV) / Infectious Mononucleosis
7. Infectious Disease: Human Immunodeficiency Virus (HIV)
8. Autoimmunity
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Schedule
Page 3
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part One:
Mechanisms of Immunity
CLS 419 Clinical Microbiology II
Serology
Page 4
MECHANISMS OF IMMUNITY
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. Differentiate innate (natural) and adaptive (acquired) immunity.
2. Compare active and passive immunity, including examples of each.
3. Describe the significance of the following factors included in the first line of defense:
a. Skin and mucous membranes
d. Environment
b. Age
e. Normal flora
c. Metabolism
f. Concomitant disease
4. Compare specific versus non-specific immunity as it relates to:
a. Phagocytosis
d. Humoral immunity
b. Inflammation
e. Memory
c. Cellular immunity
f. Opsonization
5. Differentiate humoral and cellular immunity.
6. Discuss the following components and their role in the immune system:
a. Phagocytes
f. Lymphocytes
(1) Granulocytes
(1) T cells
(2) Monocytes/macrophages
(2) B cells
b. Immunoglobulins
(3) Cytotoxic cells
c. Cytokines
(4) Natural killer cells
d. Complement
(5) Plasmacytes
e. Inflammatory mediators
7. Discuss in vivo antigen-antibody reactions.
8. Discuss complement regarding its:
a. Components
b. Activation pathways
c. Biological activities
9. Discuss the following as related to immunology:
a. Antigen
b. Antibody
(1) Complete
c. Epitope
(2) Hapten
10. Describe chemical properties of antigens.
11. Identify five physical characteristics of an effective antigen.
12. Differentiate between the following, as related to immunoglobulins:
a. Heavy and light chains
b. Constant and variable region
c. Fab and Fc fragments
13. Compare the five different types of antibody classes, including;
a. Structure
d. In vivo biological functions
b. Percent concentration in serum
(1) Complement fixation
c. Presence in secretions
(2) Placental transfer
(3) Hypersensitivity reactions
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 1: Mechanisms of Immunity
Page 5
14. Describe the sequence of events in the normal immune response.
15. Summarize the primary and secondary antibody response.
16. Differentiate between a complete and incomplete antibody.
17. Discuss the origin of cells involved in the immune system:
a. Pluripotent stem cell
b. Lymphoid progenitor
c. Myeloid progenitor
18. Compare humoral and cellular immunity, including the following;
a. Cell differentiation
b. Antigen processing and presentation
c. Function
19. Contrast T and B lymphocytes and macrophages, including:
a. Source
d. Function
b. Differentiation site
e. Response to antigen
c. Receptors
20. Differentiate between CD4 (T-helper) and CD8 (T-suppressor) cells.
21. Discuss the significance of the CD4/CD8 ratio.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 1: Mechanisms of Immunity
Page 6
Mechanisms of Immunity
Rotation II Study Questions
Multiple Choice:
1. The immunoglobulin class that binds with very high affinity to membrane receptors on basophils and
mast cells is:
a. IgG
b. IgA
c. IgD
d. IgE
2. Antibody class and antibody subclass is determined by major physiochemical differences and
antigenic variation found primarily in the:
a. Constant region of heavy chain
b. Constant region of light chain
c. Variable regions of heavy and light chains
d. Constant regions of heavy and light chains
3. Which of the following immunoglobulins is the most efficient agglutinator?
a. IgG
b. IgA
c. IgM
d. IgE
4. The complement component C3:
a. Is increased (in plasma levels) when complement activation occurs
b. Can be measured by immunoprecipitin assays
c. Releases histamine form basophils or mast cells
d. Is NOT involved in the alternate complement pathway
5. Which of the following mediators is released during T-cell activation?
a. Immunoglobulins
b. Thymosin
c. Serotonin
d. Lymphokines
6. Bence Jones proteins are:
a. Immunoglobulin catabolic fragments in the urine
b. Monoclonal light chains synthesized de novo
c. Any light chains in the urine
d. Fab fragments of a monoclonal protein
7. The J-chain is associated with which of the following immunoglobulins?
a. IgA
b. IgG
c. IgE
d. IgD
8. The presence of immune complexes indicates:
a. Polyclonal hypergammaglobulinemia
b. Inflammatory tissue injury
c. Protection from complement-dependent neutrophil chemotaxis
d. Normal host response to antigenic exposure
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 1: Mechanisms of Immunity
Page 7
9. The biologic function of the C42 complex is:
a. Cytolysis
b. To serve as a substrate of properdin factor B
c. Cleavage and activation of the C3 molecule
d. Formation and activation of the C1s molecule
10. The rapid rise, elevated level, and prolonged production of antibody following a repeat exposure to an
antigen is called:
a. Hypersensitivity
b. Arthus reaction
c. Anamnesis response
d. Primary response
11. The cells that appear to play the most significant role in the induction of tissue damage in human
immune-complex disease are:
a. Erythrocytes
b. Neutrophils
c. Monocytes
d. Lymphocytes
12. A transfusion reaction to erythrocyte antigens will activate which of the following immunopathologic
mechanisms?
a. Reaginic sensitivity
b. Arthus reaction
c. Delayed hypersensitivity
d. Complement-dependent antibody cytotoxic
13. Innate immunity can be defined as the:
a. Immunity resulting from vaccination
b. Resistance to infectious diseases acquired via subclinical infections
c. Naturally occurring defense mechanisms that provide protection form infectious agents
d. Resistance dependent on specific recognition of infectious diseases
14. The synthesis of the J-chain:
a. Occurs only in IgM-synthesizing plasma cells
b. Is a characteristic of plasma cells synthesizing dimeric and polymeric immunoglobulins
c. Is common to all lymphocytes
d. Takes place in cells other than plasma cells
15. The Arthus reaction is primarily associated with:
a. IgM antibodies
b. Mononuclear cell infiltrates
c. Complement activation by the classical pathway
d. IgE antibodies
16. Antibody to an immunogen can facilitate phagocytosis of that substance. The term used to describe
this phenomenon is:
a. Agglutination
b. Precipitation
c. Opsonization
d. Antibody affinity
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 1: Mechanisms of Immunity
Page 8
17. The human fetus has the ability to synthesize immunoglobulin. Which of the following isotypes is the
first to be synthesized during fetal development?
a. IgA
b. IgE
c. IgG
d. IgM
18. The antigen-combining site of immunoglobulin is formed by:
a. Heavy chain only
b. Light chain only
c. Constant domains of both light and heavy chains
d. Hypervariable regions of both light and heavy chains
19. The area of the immunoglobulin molecule referred to as the hinge region is located between which
domains?
a. VH and VI
b. CH1 and CH2
c. CH2 and CH3
d. CH3 and VL
20. Which of the following characteristics in NOT true of an anamnestic response compared to a primary
response?
a. Has a shorter lag phase
b. Has a longer plateau
c. Antibodies decline more gradually
d. IgM antibodies predominate
21. The host defense function(s) of monocytes-macrophages include(s):
a. Antigen presentation
b. Phagocytosis
c. Secretion of biologically active molecules
d. All of the above
e. Both A and B
22. Which of the following factors can induce fever and synthesis of acute-phase reactants?
a. Interleukin-1 and complement
b. Cachectin and interleukin-1
c. Transferrin and interleukin-1
d. Complement and interferon
23. Measurement of acute-phase proteins is useful in all of the following conditions EXCEPT:
a. Monitoring the progress of diagnosed disease activity
b. Diagnosing conditions such as myocardial infarction
c. Assessing the response to therapy in inflammatory diseases
d. Detection of complications of a know disease
24. The acute phase reactant that responds the fastest and is the most sensitive indicator of acute
inflammation is:
a. Complement
b. Ceruloplasmin
c. C-reactive protein
d. Haptoglobin
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 1: Mechanisms of Immunity
Page 9
25. A function of the cell-mediated immune response not associated with humoral immunity is:
a. Defense against viral and bacterial infection
b. Initiation of rejection of foreign tissues and tumors
c. Defense against fungal and bacterial infection
d. Antibody production
26. All of the following are a function of T cells except:
a. Mediation of delayed hypersensitivity reactions
b. Mediation of cytolytic reactions
c. Regulation of the immune response
d. Synthesis of antibody
27. Arrange the sequence of events of a typical antibody response (1-4):
____ Plateau
____ Lag phase
____ Log phase
____ Decline
28. Arrange the steps of phagocytosis in the proper sequence:
____ Digestion of bacteria
____ Increase in chemoattractants at site of tissue damage
____ Ingestion of bacteria
____ Movement of phagocytic cells
MATCHING:
29. In the blank to the left of the items in Column A, write the letter of the serological method from
Column B with which the item is associated. Methods in Column B may be used more than once.
B
A
a. Neutrophil
_____ Releases histamine and serotonin in certain
b. Eosinophil
immune reactions
c. Basophil
_____ Involved in phagocytosis and destruction of
d. Monocyte
blood borne bacteria
e. Natural killer cells
_____ Become tissue macrophages, which engulf and
digest invading microorganisms, foreign bodies,
and senescent cells within tissue spaces
_____ Destroys large parasites and modulates allergic
inflammatory responses
_____ Functions in the destruction of virally-infected
host cells
30. Match the appropriate pathway response (column A) with the pathway (column B).
A
B
_____ Activated by antigen-antibody complexes
a. Classic pathway
_____ Generates a C3 convertase
b. Alternate pathway
_____ Activated by microbial and mammalian cell
c. Both A and B
surfaces
_____ Terminates in a membrane attack complex
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 1: Mechanisms of Immunity
Page 10
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part Two:
Serological Test Methods
CLS 419 Clinical Microbiology II
Serology
Page 11
Serological Test Methods
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. Discuss the following as related to immunology testing:
a. Equivalence zone
e. Sensitivity
b. Postzone
f. Specificity
c. Prozone
g. Screening tests
d. Cross-reactivity
h. Confirmatory tests
2. Explain how the following physical factors can affect antigen-antibody reactions:
a. Ionic strength
d. Temperature
b. pH
e. Concentration ratio of antigen and antibody
c. Reaction time
3. Describe heat inactivation of patient serum, including method and purpose.
4. Contrast precipitation, agglutination, and flocculation, including:
a. Reaction time and conditions
c. Immunoglobulin class
b. Antigen state (soluble or insoluble)
d. Lattice formation
5. Explain the principle of the following immunologic methodologies, including a clinical use for each
method:
a. Agglutination/Hemagglutination
(1) Active (direct)
(3) Reverse passive (indirect)
(2) Passive (indirect)
(4) Inhibition
b. Precipitation
(1) Double immunodiffusion
(2) Immunofixation electrophoresis (IFE)
c. Flocculation
d. Neutralization
e. Fluorescent immunoassay
(1) Direct
(2) Indirect
f. Enzyme immunoassay
(1) Competitive binding (homogeneous and heterogeneous)
(2) Sandwich
(3) Fluorescent polarization immunoassay (FPIA)
(4) Microparticle capture (MEIA)
(5) Enzyme-multiplied immunoassay technique (EMIT)
(6) Chemiluminescence
g. Nephelometry
h. Western blot
i. Molecular diagnostics
(1) Polymerase chain reaction (PCR)
(3) Fluorescent in situ hybridization (FISH)
(2) Nucleic acid probes
(4) Microarray
6. Evaluate the acceptability of results, taking into account false positives and false negatives, of the
tests listed in objective #5, indicating appropriate corrective action and/or further testing if necessary.
7. Briefly explain the principle of the complement fixation test.
8. Discuss general reasons for performing immunologic/serologic testing, including:
a. Acute vs. convalescent infection
b. Prenatal vs. postnatal
c. Infectious disease serology vs. immunologic disorder
d. Immunologic testing vs. culture
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 12
Serological Test Methods
Rotation II Study Questions
Short Answer:
1. Differentiate affinity and avidity.
2. A patient’s tube agglutination titer testing for antibody in the patient’s serum gave the following
results:
Tube 1
1:2
No agglutination
Tube 2
1:4
No agglutination
Tube 3
1:8
No agglutination
Tube 4
1:16
Agglutination
Tube 5
1:32
Agglutination
Tube 6
1:64
Agglutination
Tube 7
1:128
Agglutination
Tube 8
1:256
Agglutination
Tube 9
1:512
No agglutination
Tube 10
1:1024
Negative Control, No agglutination
Describe what is taking place in this test. Are the test results acceptable? If yes, what result is
reported?
3. Name five factors (including those in the medium used) influencing antibody-antibody binding.
4. Although a high sensitivity and high specificity is desired in all tests, for screening purposes a high
________________ is more important, while for confirmation a high _____________ is more
important.
a. Sensitivity
b. Specificity
5. If the first tube in a series of one-to-two dilutions is 1:2, what is the final dilution in tube 6?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 13
6. The following serological dilutions were made:
1.8 ml of diluent was added to tube #1.
100 ul of diluent was added to tubes #2-8.
200 ul of specimen was added to tube #1. This was mixed and
100 ul was transferred to tube #2.
This same serial dilution was continued through tube #8.
What is the dilution in tube #8?
7. The following serological dilutions were made:
0.9 ml of diluent was pipetted into each of 4 tubes.
In the first tube, 0.1 ml of serum was added.
This was mixed and 0.1 ml was transferred into tube #2.
This serial dilution was done through tube #4.
What is the final dilution in tube #4?
8. You have 25 ul of a patient sample. How much diluent would you add to make a 1:100 dilution?
9. Ten ml of a 1:200 dilution is diluted to 50.0 ml. What is the final dilution? How much serum is
actually present in this final dilution?
10. How much saline and serum would you add to a tube so that it would contain 5.0 ml of a 1:250
dilution of serum?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 14
11. Calculate the following:
Dilution:
A. 1.0 ml of 1:1000 of X is diluted with 1.0 ml saline
B. 1.0 ml of “A” is diluted with 2.0 ml of saline
C. 1.0 ml of “B” is diluted with 1.0 ml of saline
D. 1.0 ml of “B” is diluted with 0.5 ml of saline
E. 1.0 ml of “C” is diluted with 3.0 ml of saline
12. Why is it important to demonstrate a four-fold rise in a specific antibody titer for presumptive evidence
of an infection??
13. A serum sample is diluted 1:5 with buffer. One (1) ml of buffer is put into each of ten tubes. A serial
dilution is made, adding 1.0 ml of the 1:5 dilution to tube one (i.e. one ml is transferred to each of the
succeeding tubes). What is the dilution in each tube?
14. How much saline and serum would you add to a tube so that it would contain 6 ml of a 1:300 dilution
of serum?
15. Which of the following influence the quality of agglutination reactions (yes or no for each):
_______ Length of incubation
_______ Amount of antigen conjugated to the carrier
_______ Avidity of antigen conjugated to the carrier
_______ Whether the carrier is artificial or biologic
_______ The pH of the system
_______ Whether agglutination or agglutination inhibition is used
16. Identify the truth about nephelometry (yes or no for each)
_______ It utilizes a modified precipitation reaction.
_______ It measures antibody-antibody concentration at equivalence.
_______ It utilizes the light-scattering properties of antibody-antibody reactions.
_______ It utilizes the light absorbing properties (turbidity) of the solution.
_______ It requires careful standardization.
_______ It requires instrumentation.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 15
Multiple Choice:
17. Immunofixation electrophoresis is best used in
a. The workup of a polyclonal gammopathy
b. The workup of a monoclonal gammopathy
c. Screening for circulating immune complexes
d. Identification of hypercomplementemia
18. The Western blot test differs from ELISA tests in which of the following ways?
a. Western blot test differs for antigen only
b. Antibodies to individual antigens can be identified in Western blot
c. Western blot detects antibody earlier than ELISA tests
d. Only ELISA testing involves enzyme labels
19. Cross reactivity in serologic reactions may lead to false positive results and can be expected when:
a. Antigens are not in optimal proportions with antibodies
b. There is no electrolyte in the test system
c. Several antigens are closely related
d. Complement has been inactivated
20. Which of the following is generally identified as important in successful function of immunoserological
assays?
a. pH above 10
b. Electrolyte-free environment
c. High-quality reagents
d. Extreme antibody excess
21. Flocculation procedures differ from latex agglutination procedures because
a. Antigen is bound to a carrier
b. Antibody is bound to a carrier
c. Soluble antigen reacts with antibody
d. They are only qualitative procedures
22. The prozone reaction may be eliminated by:
a. Incubation
b. Refrigeration
c. Dilution
d. Centrifugation
23. A postzone reaction giving a falsely negative result may occur when:
a. The quantity of antigen is excessive
b. There is more antibody than antigen
c. Serum has not been heat inactivated before titering
d. Antigens are very closely related
24. In direct immunofluorescence testing for detection of viral antigens,
a. The component fixed on the microscope slide is antiviral antibodies
b. Fluorescein-labeled antiviral antibodies bind to antigens
c. A positive (antigen present) result appears as absence of fluorescence
d. The test is performed in two stages, the second of which involves application of labeled
antihuman globulin
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 16
25. All of the following phrases describe indirect immunofluorescence testing for identification of antibody
EXCEPT:
a. A known antigen is fixed on a slide
b. A positive (antibody present) result appears as yellow-green fluorescence
c. Staining is performed in two stages
d. Fluorescein-labeled antihuman globulin attaches to known antigen used in the test system
26. All noncompetitive solid-phase enzyme immunoassays have several features in common. One of
these is:
a. One of the test components is labeled with fluorescein-isothiocyanate
b. A color change is produced by the action of complement or erythrocytes
c. An enzyme label is bound to a solid phase in the system
d. Bound enzyme acts on a substrate to produce a color change
27. In competitive enzyme immunoassays for detection of antigen,
a. A positive (antigen present) result appears as a color change
b. The antibodies on the solid phase are labeled with enzyme
c. Enzyme-labeled antigens of the same type as those being detected in the assay are incubated
with the patient’s sample
d. A simple one-step procedure is all that is required
28. Molecular diagnostics is based on which of the following concepts?
a. All viruses have unique antigens
b. Unique amino acid sequences are found in the DNA of each virus
c. Antibodies react specifically with unique determinants on the viral capsid
d. Viral replication involves unique enzymes
29. Which of the following phrases describes the polymerase chain reaction technique?
a. Is an in vitro technique for replicating viral DNA
b. Is performed by inoculating viruses into susceptible hosts to permit viral replication
c. Facilitates virus detection by attaching lengthy chains of polymers to viral surface antigens
d. Initiates a chemical chain reaction that results in production of viral enzymes that can be detected
and used to identify the virus
30. A test that is very sensitive:
a. Yields positive results only when the appropriate antibody (or antigen) is present
b. Is very delicate and requires careful quality control
c. Uses sensitized (antibody- or antigen-coated) erythrocytes as part of the indicator system
d. Detects even a very small quantity of the unknown antigen or antibody
31. In testing 100 serum samples that were known to be positive for antibody A, method 1 identified 50
samples as positive and method 2 identified 98 samples as positive. In testing 100 serum samples
that were known to be negative for antibody A, method 1 identified 100 samples as negative and
method 2 identified 88 samples as negative. Which of the following is true?
a. Method 1 is more sensitive than 2
b. Method 1 is more specific than method 2
c. Method 2 is more sensitive and more specific than method 1
d. Method 1 is more sensitive and more specific than method 2
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 17
32. Serodiagnosis of viral infections is described as an indirect diagnostic method because:
a. A two-stage procedure involving antihuman globulin is used in many of the immunoserological
procedures
b. Infecting viruses are isolated indirectly from serum rather than directly from the site of infection
c. Diagnosis relies on detection of viral antibodies in serum rather than on isolation or observation of
the infecting agent
d. Antibodies identified in serum are not type specific enough to identify the infecting virus
definitively, so such diagnosis only “indirectly” implicates the causative agent
33. In enzyme immunoassays in which the “answer” is read spectrophotometrically, an index value may
be calculated on each sample and reported to the physician. Which of the following phrases
accurately describes the index value?
a. The index value is a ratio of the absorbance values of acute and convalescent samples form a
patient; values of greater than or equal to 4 indicate a significant difference in titer.
b. The index value is a ration representing the spectrophotometric readout of the patient’s sample
divided by that of the calibrator (or cutoff value) sample; values of greater than or equal to 1
indicate a positive (antibody present) result
c. The index ratio is calculated by multiplying a sample’s absorbance value by the dilution factor
d. The index ratio is simply a sample’s absorbance reading from the spectrophotometer
34. Before performance of many serological procedures, serum specimens must be heat inactivated.
This heating:
a. Precipitates any hemolysis products that are present
b. Destroys most antibodies
c. Destroys complement and other enzymes
d. Is accomplished by incubating samples at 37ºC for 15 minutes
35. In a quantitative serological test, the results shown below were obtained. How should this result be
reported (+ = antibody detected, 0 = no antibody detected)?
Serum dilution
Test result
a.
b.
c.
d.
1:10
+
1:20
+
1:40
+
1:80
+
1:160
+
1:320
+
Report “negative” for antibody
Report “positive” for antibody
Repeat the test because this pattern is impossible
Perform testing on additional dilutions until an end point is reached
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
Page 18
MATCHING:
36. In the blank to the left of the items in Column A, write the letter of the serological method from
Column B with which the item is associated. Methods in Column B may be used more than once.
B
A
a. Complement fixation
_____ Antigen-antibody complexes fix one of the test
b. Direct (hem)agglutination
components
c. Direct immunofluorescence
_____ Soluble antigen plus specific antibody
d. Enzyme immunoassay
_____ Form of antigen is questionable (soluble versus
e. Flocculation
tiny insoluble)
f. Indirect immunofluorescence
_____ Antigen is natural part of particulate or cellular
g. Passive (hem)agglutination
antigen
h. Precipitation
_____ Bacterial cells clumped by antibody
i. Neutralization
_____ Antigen-coated particulate carrier
_____ Immunodiffusion
_____ Virus-specific antibody labeled with fluorescein
_____ Fluorescein-labeled anti-species globulin
_____ Enzyme-labeled anti-species globulin
_____ Antigen is inactivated when antibody binds to it
37. In the blank to the left of the items in Column A, write the letter of the immunoglobulin from Column B
with which the item is associated. Immunoglobulins in Column B may be used more than once.
_____
_____
_____
_____
_____
_____
_____
_____
_____
_____
A
Crosses the placenta
Found in secretions
Occurs first in infection
Appears later in infection
75% of circulating antibody
Associated with allergy and hypersensitivity
Good agglutinating, lysing and complement fixing antibody
Poor agglutinating antibody, but good in precipitation
Found on early lymphocytes
Associated with parasitic infections
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 2: Serological Test Methods
a.
b.
c.
d.
e.
B
IgG
IgA
IgM
IgD
IgE
Page 19
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part Three:
Topic 1
Infectious Diseases:
Spirochetes
CLS 419 Clinical Microbiology II
Serology
Page 20
SPIROCHETES
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. Describe the following characteristics common to the Spirochaetaceae:
a. Gram stain reaction
b. Organism morphology
c. Growth requirements
2. Discuss the three modes of transmission of syphilis.
3. Correlate the four stages of syphilis with the clinical and laboratory findings during each stage.
4. Correlate the clinical and laboratory findings associated with congenital syphilis.
5. Discuss the principle of each of the following tests, including all components and their function, as
well as the antibody being detected:
a. RPR
b. VDRL
c. FTA-ABS
d. EIA
e. DNA probe
6. Compare the tests listed in objective #5 as to their:
a. Use as a screening or confirmatory test
b. Classification as non-treponemal or treponemal test
c. Sensitivity and specificity
7. Interpret the tests listed in objective #5.
8. Distinguish between syphilis and biological false positives using RPR and/or VDRL and FTA-ABS
results.
9. List three treponemal diseases, other than syphilis, which will cause reactive treponemal testing.
10. Correlate the clinical, epidemiological and laboratory findings, including serology, associated with the
following infections:
a. Borrelia burgdorferi
1) Lyme disease
b. Borrelia species
1) Relapsing fever
c. Leptospira interrogans
1) Leptospirosis
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 21
Spirochete Diseases and Testing
Rotation II Study Questions
SYPHILIS
SHORT ANSWER:
1. What is the etiologic agent of syphilis?
2. List three modes of transmission of syphilis.
3. Describe the major symptoms / characteristics for the following stages of syphilis:
a. Primary syphilis
b. Secondary syphilis
c.
Latent syphilis
d. Tertiary syphilis
e. Congenital syphilis
4.
Which two stages of syphilis are most infectious?
5.
What disease state(s) other than syphilis can cause a true positive RPR and FTA-ABS? Why?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 22
6. How is syphilis treated?
7. Compare the two methods of direct detection of spirochetes in a specimen.
What is
How is the test
Method
Principle of test
detected in
used to diagnose
specimen?
syphilis?
Other
comments
Darkfield
microscopy
Direct
fluorescent
antibody
testing
8. Differentiate between nontreponemal serological tests and standard treponemal serological tests.
Nontreponemal Tests
Treponemal Tests
HemRPR
VDRL
FTA-ABS
agglutination
Specimen(s)
Test Principle
What is detected in
specimen?
What is the antigen
used in the test?
Screening or
Confirmatory?
Sensitivity:
Specificity:
Other Comments
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 23
9. Discuss the sensitivity of serologic tests for syphilis during primary, secondary and late stages of the
disease.
Test
RPR (screen)
Primary*
Secondary*
Latent & Tertiary*
Notes
VDRL (screen)
FTA-ABS
MHA-TP
*% sensitivity
Confirmatory tests will continue to be positive if patient is immunocompetent
10. What is the purpose of the following constituents in the RPR antigen suspension?
a. Cardiolipin
b. Choline chloride
c.
Charcoal
d. Cholesterol
11. Explain the principle of an ELISA syphilis test that only detects IgM antibody.
12. A day-old infant has the following FTA-ABS results:
FTA-ABS IgG: reactive
FTA-ABS IgM: nonreactive
Explain these results:
13. What are some biological false positives form nontreponemal serologic tests for syphilis?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 24
14. Briefly describe the principle and laboratory utilization of these newer technologies in syphilis testing:
a. ELISA
b. PCR
15. What are specific problems when performing laboratory testing to:
a. Diagnose congenital syphilis
b. Test CSF
16. Interpret the following results:
(i.e., biological false positive, primary syphilis, secondary syphilis, etc.)
Interpretation:
a. RPR: reactive
FTA-ABS: reactive
b. RPR: nonreactive
FTA-ABS: reactive
c.
RPR: nonreactive
FTA-ABS: nonreactive
d. RPR: reactive
FTA-ABS: nonreactive
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 25
Multiple Choice:
____17.
Which of the following needs to be standardized (i.e. quality controlled) when performing a
nontreponemal serologic test?
a. Needle utilized to dispense the antigen
b. Rotator speed
c. Card/slide the test is performed on
d. Antigen
e. Temperature of test system
f. All of the above
____18.
Chronic false positive RPR results are associated with which of the following?
a. Autoimmune disease
b. Chronic infections
c. Infectious mononucleosis
d. All of the above
e. A and B
____19.
A patient being seen for a routine pregnancy has a reactive RPR. What is the next best
step?
a. No further testing needed, probable false positive
b. Perform a FTA-ABS on the specimen
c. Perform a VDRL on CSF
d. Repeat testing after delivery
____20.
The following results were obtained when performing a fluorescent treponemal antibody
absorption test:
Reactive-PBS
Reactive
(4+)
Reactive-sorbent
Reactive
(4+)
Nonspecific-PBS
Reactive
(3+)
Nonspecific-sorbent
Reactive
(2+)
Minimally reactive
Reactive
(1+)
Patient sample
Reactive
(1+)
Your next step would be to:
a. Report the FTA-ABS as positive and perform a quantitative RPR
b. Report the FTA-ABS as minimally reactive and perform a quantitative RPR
c. Repeat the test because the control results were unsatisfactory
d. Request a new specimen because patient was minimally reactive
____21.
Nontreponemal serologic tests for syphilis are utilized as screening tests because:
a. Treponemal serologic tests aren’t positive during primary syphilis
b. These tests are simpler (easier technically and quicker to perform) and less expensive
than the treponemal serologic tests
c. Serum need not be heat inactivated prior to testing, while serum for treponemal serologic
testing needs to be heat inactivated.
d. All of the above
____22.
A serum reported as “reactive” for the RPR test should be confirmed by a treponemal
serologic test because nontreponemal tests are:
a. Subject to false positives
b. Not sensitive
c. Not standardized for testing serum
d. All of the above
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 26
Lyme’s Disease
SHORT ANSWER:
23. Identify and list characteristics of the causative agent of Lyme disease.
24. How do humans acquire Lyme disease?
25. Describe the major characteristics the following stages of Lyme’s disease:
a. Localized rash
b. Dissemination to multiple organ systems
c.
Chronic state
26. Briefly describe laboratory detection of Lyme disease.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 27
Multiple Choice:
____27.
Laboratory diagnosis of Lyme disease is most often done by:
a.
b.
c.
d.
____28.
After the initial infection, a detectable IgM response to Borrelia burgdorferi occurs in:
a.
b.
c.
d.
____29.
Darkfield microscopy of blood and CSF.
Detection of RF in synovial fluid
Isolate/culture Borrelia burgdorferi in blood culture medium
Serology - detection of antibodies to in serum
3-6 days
3-6 weeks
3-6 months
Never, a cellular (T-cell) is the primary immune response
Serologic tests (IFA and ELISA) for Lyme disease lack specificity because of:
a. Low levels of specific antibody produced in the early stages of the disease.
b. Cross-reactivity with other treponemal diseases (syphilis, yaws, relapsing fever,
leptospirosis)
c. High incidence of prozone
d. Increased incidence of C-reactive protein and RF in sera
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Spirochetes
Page 28
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part Three:
Topic 2
Infectious Diseases:
Streptococcal
CLS 419 Clinical Microbiology II
Serology
Page 29
STREPTOCOCCAL ANTIBODY TESTING
Objectives: Upon completion of this unit of CLS 419, the Clinical Laboratory Science student will be able
to:
1. Discuss the two delayed sequelae that can result from a Group A Streptococcus infection.
2. Compare the five antigenically significant Group A Streptococcus extracellular products, including the
importance of performing tests for antibodies to these antigens.
3. Explain the principle of:
a. Streptozyme hemagglutination test
b. Anti-DNase B neutralization test
c. Anti-Streptolysin O (ASO) latex agglutination test
4. Correlate epidemiological, clinical and laboratory findings with:
a. Group A streptococcal infection
b. Acute glomerulonephritis
c. Rheumatic fever
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Streptococcal
Page 30
Streptococcal Antibody Testing
I.
Classification of Streptococci by Lancefield typing
A serologic way to classify the Streptococci (gram-positive cocci) by detecting the “C” carbohydrate
substance that is unique to a particular Streptococcus sp. or a group of Streptococci
For example:
Group A Streptococcus = Streptococcus pyogenes
Group B Streptococcus = Streptococcus agalactiae
II. Examples of Diseases Caused by Group A Streptococcus
A. Suppurative - organism is present
1. Pharyngitis: “Strep Throat,” classic form of disease is acute, febrile and self-limiting
2. Scarlet Fever: pharyngitis with rash due to organism producing erythrogenic toxin
3. Impetigo: superficial skin infection with crusted lesions, very contagious, often in combination
with Staphylococcus aureus
4. Erysipelas: infection of deeper layers of the skin not involving the surface, involves
lymphatics
5. Virulence factor = M protein: a cell protein found in association with hyaluronic capsule, if
organism doesn’t produce M protein, avirulent
a. Inhibits phagocytosis
b. Antigenic
B. Non-suppurative (delayed sequelae, organism NOT present)
Two types:
Acute glomerulonephritis (occurs in up to 28% of Group A Strep infections)
Rheumatic fever (occurs in 2-3% of Group A Strep pharyngeal infections)
1. Acute glomerulonephritis
a. Sequelae of either pharyngitis (latent period 10 days) or cutaneous infections (latent
period up to 6 weeks)
b. Acute inflammatory disease of the glomerulus
c. Major clinical manifestations include:
Gross hematuria
Proteinuria
Edema of face and legs
Hypertension
Reduced levels of C3
Renal impairment
d. More predominant in males
e. Pathogenesis mechanism unknown, possible reasons:
i. Cross-reactive antibodies induced by streptococcal antigens
ii. Immune complexes that accumulate in the glomeruli
2. Rheumatic fever
a. Sequelae of only pharyngitis (latent period is 18 days)
b. Inflammatory process that may involve the heart, joints, subcutaneous tissues and central
nervous system. Symptoms include polyarthritis, carditis, chorea (muscle weakness with
quick, involuntary, purposeless movements and personality changes), and erythema
margination (cutaneous rash).
c. 30-60% of effected individuals suffer permanent disability
d. Pathogenesis mechanism unknown, but thought to be antibodies formed against
streptococcal antigens cross-react with skeletal and smooth muscle of vessel walls
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Streptococcal
Page 31
III. Extracellular Antigens Produced by Group A Streptococcus - produces more than 20 different
exotoxins
A. Streptolysin O
1. Lyses RBCs
2. Very antigenic
3. Oxygen labile
4. Neutralized by anti-streptolysin O
5. Inhibited by cholesterol, detergents, reducing substances, oxidizing agents and bacteria
B. Streptolysin S
1. Lyses RBCs
2. Not antigenic
3. Oxygen stable
4. Not inhibited by cholesterol
C. DNase-B
1. Depolymerizes DNA
2. Antigenic
3. Antibody titers are of great value in the diagnosis of pharyngeal or skin infections
D. Streptokinase
1. Dissolves fibrin clot (may serve as spreading factor)
2. Antigenic
E. Hyaluronidase
1. Splits hyaluronic acid (hyaluronic acid is a component in collagen which is in connective
tissue)
2. Antigenic
F. NADase
1. Splits nucleotides - facilitates invasiveness of organism
2. Antigenic
G. Antibiotic therapy may suppress the production of antibody to the above antigens.
IV. Diagnostic Serologic Tests For Diagnosis of Group A Streptococcus Non-suppurative Infections
A. Streptozyme Test
1. Passive hemagglutination test
2. Antigens coated on sheep RBCs
a. Streptolysin O
b. Streptokinase
c. Hyaluronidase
d. DNase
e. NADase
3. Screening test
B. Anti-streptolysin O (ASO) Test
1. Detects antibodies to Streptolysin O
2. Neutralization Test: classic method, titer = highest dilution that neutralizes the streptolysin O
antigen which lyses RBCs
Units = Todd Units
Normal = < 166 Todd Units
3. Currently: latex agglutination screening test
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Streptococcal
Page 32
C. Anti-DNase B Titer
1. Neutralization Procedure
Serum + DNase B, incubate; then add DNA indicator
Toluidine Blue + Free DNA = blue color (pink color = no free DNA)
If serum positive for antibody to DNase B (or anti-DNase B), the DNase B in the first
incubation step will be neutralized. When the DNA is added, the DNase-B will be neutralized
and thus unable to break down the DNA. The free DNA is unchanged and the indicator is
blue.
If serum is negative for anti-DNase B, the DNase B in first incubation step is unaltered. It is
then able to breakdown the DNA in the second step. There is no free DNA and the indicator
turns pink.
2. These antibodies rise later than ASO
3. Titers remain higher for a longer period of time than ASO
4. More frequently positive with acute glomerulonephritis due to skin infections
5. Considered to be the single best test for the serologic detection of Group A
Streptococcal Infections
D. Acute glomerulonephritis
1. Urinalysis
2. Streptozyme Test
3. Anti-DNase B (preferred) or ASO
E. Rheumatic Fever
1. Streptozyme Test
2. ASO
3. Anti-DNase B
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Streptococcal
Page 33
Streptococcal Antibody Testing
Rotation II Study Questions
Multiple Choice:
1. A patient's ASO titer, using a latex agglutination procedure, is 200 U/ml units. This most likely
represents which of the following?
a. A recent infection with streptococci
b. A current infection with streptococci
c. Not able to determine with a single titer
2. The Streptozyme test screens for which of the following antibodies?
a. Anti-streptolysin O
b. Anti-DNase
c. Anti-NADase
d. Anti-hyaluronidase
e. All of the above
3. A positive Streptozyme test shows:
a. Agglutination
b. Hemolysis
c. No agglutination
d. Both a and b
4. A positive Streptozyme and a negative ASO latex test might be explained by:
a. Heat inactivated serum was tested
b. The presence of antibodies to DNase B, streptokinase and hyaluronidase only
c. The presence of antibodies to streptolysin-O at a maximum titer of 200 U/ml
d. The absence of an acute streptococcal infection in the patient
5. Long-term complications of Streptococcus pyogenes infection include:
a. Acute rheumatic fever
b. Post-streptococcal glomerulonephritis
c. Rheumatoid arthritis
d. Both A and B
Short Answer:
6. Describe the methodologies used in the following tests. Identify what antibodies are being tested for.
a. Streptozyme:
b. ASO latex agglutination:
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Streptococcal
Page 34
c.
Anti-DNase B titer:
7. Overall, which of the above tests/antibody detections is the most sensitive and specific in detecting a
recent Group A Streptococcus infection? Why?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases - Streptococcal
Page 35
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part Three:
Topic 3
Infectious Diseases:
Viral & Parasitic
CLS 419 Clinical Microbiology II
Serology
Page 36
EPSTEIN-BARR VIRUS (EBV)/INFECTIOUS MONONUCLEOSIS
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. Identify the etiologic agent of infectious mononucleosis.
2. Describe the clinical symptoms of infectious mononucleosis.
3. Explain the principle of presumptive and differential testing for infectious mononucleosis antibodies.
4. Differentiate infectious mononucleosis heterophile antibodies from serum sickness and Forssman
heterophile antibodies when using the Davidsohn differential test or a modification of that test.
5. Correlate heterophile antibody test results with hematology findings associated with infectious
mononucleosis.
6. Chart the appearance and disappearance of the following EBV and heterophile antibodies during the
course EBV infection, chronologically:
a. Viral capsid antigen (VCA) IgM
b. Viral capsid antigen (VCA) IgG
c. Early antigen- diffuse (EA-D)
d. Early antigen- restricted (EA-R)
e. Epstein Barr nuclear antigen IgG (EPNA)
f. Heterophile
7. Correlate the clinical symptoms, pathophysiology, and laboratory findings in infections
mononucleosis.
MISCELLANEOUS SEROLOGY TESTING
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. List the components of the TORCH panel.
2. Describe population groups that should be screened for immunity to Rubella.
3. Correlate the clinical significance, epidemiology and laboratory findings associated with the following
infections:
a. Toxoplasma gondii
b. Rubella
c. Cytomegalovirus
d. Herpes Simplex I and II
e. West Nile virus
4. Correlate the clinical and laboratory findings associated with congenital infections for each of the
organisms listed in Objective #3.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 37
Immunological Testing for Viral Infections
I.
Rubella
A. Etiology – Rubella Virus
1. Enveloped, single stranded RNA III
B. Epidemiology Originally called “third disease”
1. Transmitted by
a. Respiratory secretions
b. Transplacental transfer
2. Populations that require immune status
a. Preschool and school-aged children
b. OB / all females at or just before childbearing age
c. Premarital women (many states no longer require this)
d. School age boys (6-9 years)
e. Healthcare personnel
3. Vaccination
a. Utilizes live virus
b. Should not be used during pregnancy (due to live virus)
C. Symptoms
1. Postnatal or acquired infection
a. Incubation period of 10-21 days
b. Fever >100ºC
c. Macropapular rash
2. Congenital infection
a. Severe in first trimester
b. Effects CNS, eyes, heart, bone; can cause deafness, hepatomegaly, splenomegaly,
seizures, and microcephaly
D. Immunological Manifestation Infection with Rubella usually results in lifelong immunity
E. Diagnostic Methodologies
Part of the TORCH panel of tests (Toxoplasma, Rubella, CMV, Herpes)
1. Hemagglutination Inhibition (reference method)
2. EIA (IgG immune status, IgM acute infection)
II. Cytomegalovirus (CMV)
A. Etiology – member of herpes virus group
B. Epidemiology
1. Transmitted by
a. Oral, respiratory or venereal
b. May be transmitted by organ transplantation or blood transfusion
2. Endemic worldwide
3. A cell-associated virus (i.e. culture from cellular material, not fluid)
C. Symptoms
1. Postnatal acquired infections In all populations the virus alters the immune system that
decreases the immune response to the virus
2. Health adults
a. Usually asymptomatic
b. Mono-like illness
3. Immunosuppressed adults
a. Can be life threatening
b. May result in disseminated multi-system involvement
c. Patient is treated
4. Infants
a. Transmitted via cervical secretions, blood or breast milk
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 38
5. Congenital
a. Most common intrauterine infection 0.4-2.0% of all live births
b. If mother has Ab to CMV before conception, baby is at much lower risk for developing
severe disease
c. Mild, asymptomatic, jaundice
d. Severe, Cytomegalic Inclusion Disease resulting in neurologic and neuromuscular
disorders, hepatomegaly, splenomegaly
D. Diagnostic Methodologies
Part of the TORCH panel of tests (Toxoplasma, Rubella, CMV, Herpes)
1. Isolation / Culture of virus from
a. Urine (CMV persists for months)
b. Nasopharynx (swabs, secretions)
c. Blood (culture buffy coat)
2. Conventional Viral Culture
a. Monolayer of diploid human fibroblast cells inoculated with specimen
b. 3-6 weeks for growth
3. Rapid shell vial technique
a. Grow human fibroblast cells on a coverslip
b. Inoculate with specimen and centrifuge
c. Incubate
d. Stain with monoclonal antibody
4. Serology
a. High incidence in the general population (60-90%)
b. Methodologies
i. Complement fixation
ii. IFA
iii. EIA (serum)
III. Herpes
A. Etiology – DNA virus in Family Herpesviridae – double-stranded DNA
B. Epidemiology
1. Herpes Simplex Virus I (HSV I) - oral
2. Herpes Simplex Virus II (HSV II) - genital
C. Symptoms
1. Primary infection
a. Lesions
b. Fever
c. Lymphadenopathy
2. Latency
3. Recurrences
4. Congenital
a. Causes more severe disease in newborns
b. Intrauterine infection
i. Primary genital infection in mother
ii. Recurrent infection in mother
c. Symptoms at birth
i. Vesicular skin rash
ii. 80% brain damage
d. Transmission during delivery (more common)
D. Diagnostic Methodologies
Part of the TORCH panel of tests (Toxoplasma, Rubella, CMV, Herpes)
1. DFA Direct smear of vesicle fluid
2. Culture 1-7 days, will distinguish between HSV I and HSV II
3. EIA Looking for Ab, does not distinguish between HSV I and HSV II
4. Nucleic Acid Detection (PCR) Looking for organism
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 39
IV. Epstein-Barr Virus (EBV)
A. Etiology
1. DNA virus in Family Herpesviridae
2. Infects B-lymphocytes
3. Primary cause of IM (90% of IM attributed to EBV, 6% of IM cases caused by
Cytomegalovirus - CMV)
4. Malignancies/Cancers
a. B-lymphocyte lineage tumors: Burkett’s lymphoma and primary lymphoma of the brain
b. Nasopharyngeal carcinomas (epithelial cancer)
c. Gastric carcinomas
d. Neoplasms of thymus, parotid gland and larynx
5. Neurologic syndromes: Bell’s palsy, Guillian-Barré syndrome, meningoencephalitis, Reye’s
syndrome
6. HIV infected individuals: associated with diffuse polyclonal lymphomas, pneumonitis, oral
hairy leukoplakia of tongue
7. X-linked Lymphoproliferative Syndrome (XLP) - a genetically linked, lymphoma-like disease
in young boys following infectious mononucleosis (often fatal)
8. Reactivation of latent infection has been “implicated” in chronic fatigue syndrome (not
universally accepted)
B. Epidemiology
1. The most ubiquitous virus known to man
a. 50% of population exposed to EBV by 5 years
b. 90% of adults demonstrate antibodies to EBV
2. Transmission
a. Close contact: oral-pharyngeal secretions of infected individual (infectious during
incubation period and up to 5 mo. after recovery)
b. Rarely: blood transfusion, transplacental transfer
3. Immunity After primary exposure, a person is considered immune and no longer susceptible
to overt reinfection (with normal immune function)
4. Incubation period 10-50 days; if symptomatic lasts from 1-4 weeks
C. Symptoms
1. Usually asymptomatic when primary infection occurs in early childhood
2. Symptoms usually occur when primary infection occurs at 10-20 years of age, in high
socioeconomic groups who typically are not infected early in life
3. Clinical manifestations
a. Extreme fatigue and malaise
b. Lymphadenopathy (particularly in posterior cervical area)
c. Fever
d. Sore throat
e. Splenomegaly (50%)
f. Hepatomegaly - because abnormal liver function is more apparent with EBV IM, EBV
must be considered in the differential diagnosis of hepatitis
4. Prognosis
a. Usually complete recovery after a few weeks
b. Occasional relapses (5-10%)
c. Life-long immunity (with normal immune system)
d. Rare complications or death (rupture of spleen, myocarditis, aseptic meningitis,
encephalitis)
D. Diagnostic Methodologies Three criteria:
1. Characteristic clinical signs & symptoms (see above)
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 40
2. Characteristic hematological picture
a. Moderate increased WBC count
b. > 50% lymphocytes
c. 10-20% atypical lymphs
d. EBV infects B lymphocytes and then infects other lymphocytes, remains latent in B
lymphocytes
e. T-cells are activated which eventually eliminate the infected B-cells and symptoms occur.
f. Atypical lymphocytes appear to be activated T-cells
3. Positive IM serology
a. Nonspecific tests: detect presence of nonspecific IM heterophile antibodies
b. Specific tests: detect presence of specific EBV antibodies
c. General principles of heterophile antibody testing:
Heterophile antibodies are a group of nonspecific antibodies. These antibodies are
stimulated by one antigen and react with an entirely unrelated antigen present on cells
from different mammalian species.
Examples of heterophile antibodies:
ƒ IM
ƒ Forssman (stimulated by various antigens in nature)
ƒ Serum Sickness (allergic reaction to large doses of animal protein)
ƒ All three of these heterophile antibodies will agglutinate ox, sheep or horse RBCs
d. IM heterophile antibodies:
i. React with:
a) Ox, sheep, horse RBCs (agglutinate)
b) Beef RBCs (absorbed)
ii. Do not react with:
a) Guinea pig kidney cells
b) EBV-specific antigens
iii. Positive test
a) Usually positive by 6th - 10th day of onset of symptoms
b) Highest titer = 2nd to 3rd week, persist for minimum of 6 weeks
c) Level of antibody does not correlate with severity of disease
iv. Sero-negative (negative for IM heterophile antibodies) IM
a) False negative if tested before IM heterophile antibody response is at a high
enough titer to be detected
b) 10-20% of individuals with acute IM do not produce IM heterophile antibody
e. Serologic Tests for Heterophile Antibodies
i. Paul-Bunnell Screening Test - detects the presence of IM, Forssman and Serum
Sickness heterophile antibodies with no differentiation
Patient serum + 2% suspension washed sheep RBCs, mix:
Agglutination = (+) for heterophile Ab
No agglutination = (-) for heterophile Ab
ii. Differential Screening Tests - Davidsohn Differential / Monospot and other
modifications of Davidsohn Differential test
Serum is mixed with guinea pig kidney.
This mixture is then mixed with horse RBCs.
If the heterophile antibody present is Forssman or Serum Sickness, the antibody will
be absorbed out with the guinea pig kidney (i.e., reacts with) and will be “used up”
and unable to react with or agglutinate the horse RBCs.
The IM heterophile antibody will not react with the guinea pig kidney and will be
available to react with or agglutinate the horse RBCs.
Davidsohn Absorption Patterns
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 41
Type of Heterophile
Ab
IM
Forssman
Serum Sickness
f.
Absorbed by guinea pig kidney
cells
Absorbed by beef
RBCs
No
Yes
Yes
Yes
Yes (partially)
Yes (completely)
Serum + horse RBCs = agglutination (positive for a heterophile antibody)
Serum + guinea pig kidney, mix
Serum/guinea pig kidney mixture + horse RBCs, mix:
Agglutination: positive for IM heterophile antibody
No agglutination: negative for IM heterophile antibody
Serologic Tests for Specific EBV Antibodies
i. Purpose: To confirm infectious mononucleosis in:
a) Heterophile negative patients (<10%)
b) Immunosuppressed or patients who do not exhibit classic symptoms
ii. Antigens
a) Viral Capsid Antigen (VCA): produced by and found in cytoplasm of infected Bcells
• Anti-VCA IgM is usually detected in low concentrations early in the infection
and disappears within 2-4 months.
• Anti-VCA IgG is usually detected within 4-7 days after the onset of
symptoms and persists for an extended time, perhaps lifetime.
b) Early Antigen (EA)
• Diffuse (EA-D): Found in nucleus and cytoplasm of infected B-cells
IgG-EA-D is highly indicative of acute infection but is detectable in only 1020% of IM patients (i.e., specific but not sensitive)
It disappears in approximately 3 months with a rise in titer during reactivation
of latent infections.
• Restricted (EA-R): Found as a mass in the cytoplasm of infected B-cells
IgG-EA-R sometimes demonstrated in serum of very young children during
the acute phase.
IgG-EA-R appears transiently in the later convalescent phase.
c) Epstein-Barr Nuclear Antigen (EBNA): Found in the nucleus of all EBV infected
cells
• Not available for antibody stimulation until the T-lymphocytes destroy the Bcells. Therefore, anti-EBNA is absent or barely detectable during acute IM.
• Anti-EBNA IgG does not appear until the patient has entered the
convalescent period. Antibody titers gradually increase for 3-12 months postinfection and remain raised indefinitely due to the persistent viral carrier
state.
iii. Methodologies
a) ELISA
b) Immunofluorescence
4. Clinical Correlations (see Table 17-3, page 269, Stevens textbook)
a. Organisms causing similar clinical symptoms, but are differentiated by EBV serology or
hematological picture:
Cytomegalovirus, Adenovirus, Rubella, Toxoplasma, Herpes, Viral Hepatitis, Group A
Streptococcus
b. Conditions causing similar hematological picture as IM, but differentiated by EBV
serology and clinical symptoms:
Acute lymphocytic leukemia, Cytomegalovirus, Drug reactions, Serum sickness
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 42
c.
Other antibodies sometimes present with IM:
Cold agglutinins, Biological false positive RPR, Anti-IgG resembling RF, Speckled
ANA, Platelet and WBC antibodies
VCA
IgG
EBNA
IgG
No previous exposure
neg
Neg
neg
neg
neg
Heterophile
neg
Recent (acute) infection
pos
Pos
pos/neg
neg
neg
pos
Past infection (convalescent)
period
neg
Pos
neg
neg
pos
neg
Reactivation of latent
infection
pos /
neg
Pos
pos /
neg
pos /
neg
pos
pos/neg
VCA IgM
EA-D
EA-R
VCA = Viral capsid antigen
EA-D = Early antigen (diffuse)
EA-R = Early antigen (restricted)
EBNA = Epstein-Barr nuclear antigen
V. Hepatitis
A. Etiology – Hepatitis A, B, C, D, and E viruses
B. Refer to Chemistry notes
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 43
Immunological Testing for Parasitic Infections
I.
Toxoplasmosis
A. Etiology – Toxoplasma gondii
B. Epidemiology
1. Protozoan parasite of cosmopolitan distribution
2. Definitive host: house cat
3. Intermediate hosts
a. Mice
b. Dogs
c. Humans
d. Cows
e. Pigs
f. Birds
g. Other mammals
4. Transmitted by:
a. Ingestion of oocysts passed in feces of definitive host via contaminated food and water
b. Ingestion of oocysts in undercooked meat of intermediate host
c. Transplacental transmission Usually takes place in the course of an acute but unapparent
or undiagnosed maternal infection
C. Symptoms
1. Usually asymptomatic
2. Chronic problem in immunosuppressed
D. Congenital Infection
1. Disease is more severe the earlier it is acquired during pregnancy
2. Can cross the placenta
E. Diagnostic Methodologies
Part of the TORCH panel of tests (Toxoplasma, Rubella, CMV, Herpes)
1. EIA / ELISA
a. Procedure
i. Diluted patient serum (Ab) is added to antigen (Ag) wells. If specific Toxoplasma Ab
is present, it will bind to the Ag
ii. Unbound Ab is washed away
iii. Enzyme-conjugated antihuman IgG is added and binds to the Ag-Ab complex
iv. Excess enzyme conjugated AHG is washed away
v. Substrate is added and the bound enzyme conjugate begins a hydrolytic reaction
vi. After a specified time, the reaction is stopped and read by a spectrophotometer
b. Interferences
i. False positives: RF
ii. False negatives: too early in disease (Ab detectable 2 weeks after onset)
2. Sabin-Feldman Dye Test
a. Not used today because it is hazardous and difficult to perform
b. Live Toxoplasma gondii organisms are incubated with patient serum. If Ab to Toxoplasma
is present, the organism is killed
c. Methylene blue stain is added
d. Only live organisms (NOT destroyed by patient Ab) will stain; therefore live organisms are
interpreted as a negative result
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 44
Immunological Testing for Viral Infections (including TORCH)
Rotation II Study Questions
Multiple Choice:
1. In a woman of childbearing age, which of the following constitutes valid evidence of rubella immunity?
a. The patient says she had rubella as a child
b. The patient says she has been vaccinated against rubella
c. Her physician knows she has experienced the classic presentation of rubella
d. Serologic confirmation of past rubella infection
2. The Rubella virus is a:
a. Cytomegalovirus
b. Togavirus
c. Herpes virus
d. Retrovirus
3. A pregnant woman was exposed to a child with rubella. Hemagglutination inhibition tests on acute
and convalescent serum revealed that she had contracted rubella. Which of the following pairs of
titers best represents this scenario?
Acute Convalescent
a. 1:128 1:128
b. 1:16 1:128
c. 1:128 1:16
d. 1:32 1:64
4. During a prenatal exam an expectant mother has a positive IgG rubella antibody titer. After the birth
of the baby, testing revealed the infant also had a positive IgG rubella antibody titer. Which of the
following statements is most likely true concerning the infant’s results?
a. The infant probably has rubella
b. The mother passed the rubella virus to the infant
c. More testing would need to be done to evaluate properly
d. The samples were mixed up
5. An 8-year-old boy was tested to determine whether the rash he experienced recently was due to
rubella. One serum sample was collected 4 days after the rash appeared, and a second sample was
collected 14 days later. Results of a traditional rubella antibody test (which detects primarily IgG)
were as follows:
Sample 1 = <1:10
Sample 2 = 1:160
What do these results indicate about the cause of the rash?
a. The results are inconclusive
b. Several additional samples collected in a 6-month period should be tested
c. The rash was due to rubella infection
d. The boy was immune to rubella before the rash
6. A pregnant woman was tested for rubella antibodies (using a traditional rubella antibody test that
detects primarily IgG) because she thought she had been exposed. Her sample was collected within
10 days of exposure, and her rubella titer was 1:32. What do these results indicate?
a. She was immune before exposure
b. She has active rubella presently
c. A second sample is required to evaluate results
d. She has not been exposed to or immunized against rubella
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 45
7. An infant was tested for rubella antibodies (using a traditional rubella antibody test that detects
primarily IgG) at birth and at age 6 months. His results were as follows:
At birth = 1:160
At 6 months = <1:10
What do these results indicate?
a. He did not have congenital rubella
b. He was exposed to rubella shortly after he was born
c. He is probably immunodeficient
d. Data are insufficient to make a diagnosis
8. Consider the following rubella antibody testing results form tests for rubella IgM and IgG:
At infant’s birth
3 mo after birth
6 mo after birth
Patient
IgG
IgM
IgG
IgM
IgG
IgM
Mother
1:128 1:10
1:64
<1:10
1:64
<1:10
Infant
1:128 1:40
1:256 1:20
1:512 1:10
What do these results suggest?
a. The infant probably has a congenitally acquired rubella infection
b. The infant was born with something in his serum that gave false-positive results in the rubella IgM
test
c. The infant was infected by rubella at age 3 months
d. The infant’s high rubella titer at 6 months is due to maternal antibody
9. All of the following descriptive characteristics are true of cytomegalovirus EXCEPT:
a. A herpes family virus
b. A DNA virus
c. A cell-associated virus
d. Is epidemic worldwide
10. Cytomegalovirus (CMV) is associated with all of the following clinical syndromes EXCEPT:
a. Congenital infection in infants whose mothers experienced primary CMV infections during
pregnancy
b. An infectious mononucleosis-like syndrome in adolescents and young adults
c. Serious, often fatal, pneumonia in immunocompromised individuals, especially bone marrow
transplant recipients
d. Painful genital lesions in individuals with multiple sex partners
11. You are working with a sample from a lip lesion. Herpes simplex virus (HSV) is suspected. What
information is accurate concerning availability and timeliness of HSV laboratory testing?
a. HSV grows slowly in cell culture; so do not expect a positive result before 7-10 days
b. Tests for HSV antigen detection are not widely available, so virus isolation is the test of choice
c. The lesion sample can be tested for HSV antigen and inoculated into cell cultures. Antigen tests
are widely available and HSV grows quickly in culture.
d. A serum sample, rather than the lip lesion sample, should be collected and tested for HSV
antibodies. If antibodies are present, the patient is immune to HSV and the lip lesion cannot be a
result of HSV infection.
Short Answer:
12. What diseases can a TORCH screen help to diagnose?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 46
13. Describe how rubella can be transmitted.
14 List six groups of people who should be screened for rubella. Which of these groups is most
frequently assayed for active rubella infection? Why?
15. Briefly explain the possible effects of the rubella virus on a fetus. In which stage of pregnancy is
infection typically more severe?
16. Discuss the transmission of Cytomegalovirus.
17. List at least 5 body sites that CMV can be isolated from.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 47
18. Briefly describe the ELISA test as used in the CMV antibody detection test.
19. List two routes of congenital herpes infection.
20. What methodology(ies) is/are used in testing for West Nile Virus (WNV)?
21. What specimen(s) is/are used when testing humans for WNV?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 48
Immunological Testing for Parasitic Infections (including TORCH)
Rotation II Study Questions
Multiple Choice:
1. A 2-day old infant was tested for Toxoplasma gondii with the following results:
Toxo IgG = increased
Toxo IgM = negative
The most likely cause is:
a. A recent infection with Toxoplasma gondii
b. A current infection with Toxoplasma gondii
c. No infection with Toxoplasma gondii
d. A chronic infection with Toxoplasma gondii
2. Which of the following concerning Toxoplasma gondii is false?
a. A retrovirus
b. Can cause ocular lesions
c. Felines are a frequent host
d. Can cause severe central nervous system abnormalities in newborns
e. Can be acquired in utero
3. What could be the cause of the false negative in Toxoplasma IgM antibody testing?
a. Rheumatoid factor
b. High Toxoplasma IgG antibody levels
c. Serum was heat inactivated
d. Lipemia
4. All of the following are specific methods for preventing congenital toxoplasmosis EXCEPT:
a. Avoid touching mucous membranes while handling raw meat
b. Wash hands thoroughly after handling raw meat
c. Eliminate food contamination by flies, cockroaches, and other insects
d. Dispose of fecally contaminated cat litter into plastic garbage bags
5. The presence of IgM to T. gondii in an adult is indicative of
a. Carrier state
b. Active infection
c. Chronic infection
d. Latent disease
Short Answer
6. Congenital toxoplasmosis exhibits the most serious effects if the disease is contracted in the
_______________ trimester.
7. Discuss how toxoplasmosis can be transmitted.
8.
Briefly describe the ELISA test as used in the Toxoplasma antibody detection test.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – Viral & Parasitic
Page 49
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part Three:
Topic 4
Infectious Diseases:
HIV
CLS 419 Clinical Microbiology II
Serology
Page 50
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. Explain why HIV is classified as a retrovirus.
2. Discuss the epidemiology of HIV infection, including:
a. Modes of transmission
b. High risk groups
c. Prevention
3. Discuss the impact of HIV upon the immune system in regards to:
a. T-helper cells (T4, CD4)
b. T-suppressor cells (T8, CD8)
c. Interleukin-2 (IL-2)
d. Natural killer cells (NK cells)
e. B cells and immunoglobulin response
f. Cytotoxic T cells
g. Antigen-presenting cells
4. Describe the antibody response to the following antigens utilized in HIV serologic testing:
a. gp41
b. gp120/160
c. p24
5. Explain the principle of the following tests utilized in HIV serologic testing, including specificity,
sensitivity and interpretation of results:
a. Enzyme immunoassay (EIA)
b. Western blot
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 51
Human Immunodeficiency Virus (HIV)
I.
Introduction
A. Nomenclature
1. Human Immunodeficiency Virus (HIV)
a. HIV-1
b. HIV-2
2. Retrovirus: carries a single RNA strand and uses a special enzyme, reverse transcriptase, to
convert viral RNA into DNA (usually DNA is converted to RNA)
II. HIV-1
A. Structure: icosahedral with 72 external spikes
B. Contains 3 genes that encode for proteins
1. Group-specific antigen/core proteins (gag) are part of the nucleocapsid providing stability for
the shell (i.e., p18, p24, p55 )
2. Polymerase proteins (pol) are enzymatic proteins such as reverse transcriptase (i.e., p31,
p51, p66)
3. Envelope proteins (env) are envelope glycoproteins that are responsible for adhering to
target cells (i.e., gp41, gp120, gp160)
C. Life Cycle
1. gp120 binds to the surface of a cell which has CD4 receptor (i.e. T-helper cell)
2. Viral core is injected into the host cell
3. Enzyme reverse transcriptase converts the viral RNA into proviral DNA
4. DNA migrates to the cell’s nucleus where it is incorporated into the cell’s genome
5. Some of the RNA developed will remain in the host cell and help develop new virus particles
6. Transcription and protein synthesis
7. Assembly of viral proteins and RNA at cell surface
8. Mature virion “buds” from host cell
D. Transmission
1. These body fluids have been proven to spread HIV:
a. Blood and other body fluids containing blood
b. Semen
c. Vaginal fluid
d. Breast milk
2. These are additional body fluids that may transmit the virus that health care workers may
come into contact with:
a. Cerebrospinal fluid (CSF)
b. Synovial fluid
c. Amniotic fluid
3. Other isolated body fluids NOT implicated in transmission
a. Saliva
b. Tears
c. Sweat
d. Urine
e. Feces
4. Casual contact - NO
5. Survival outside the host is limited
6. Mechanisms of Transmission
a. Sexual
b. Blood
c. Congenital
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 52
7. Groups at risk
a. Homosexuals
b. IV drug users
c. Hemophiliacs
d. Sexually active not practicing safe sex
e. Other identifiable groups
i. Sexual partners of drug addicts
ii. Children born to HIV positive women
iii. Heterosexuals
iv. Health care workers
III. Clinical Diagnosis
No clear defined clinical staging system has been generally accepted. Categories in reality have
indistinct borders with much overlap and the clinical status associated with each may vary greatly on
an individual basis.
CDC Classification System
A. Group I: Acute HIV Syndrome
From time of infection to detection of antibody response, typically 12 to 20 weeks, CD4 + T-cell
counts initially drop and then rise to normal levels (500-1300 cells/mm3), plasma viremia with
wide dissemination of virus
Clinical Findings: fever, pharyngitis, lymphadenopathy, headache/retro-orbital pain, arthralgia,
lethargy, anorexia, nausea, vomiting, diarrhea
Possible: meningitis, encephalitis, erythematous maculopapular rash, mucocutaneous ulceration
B. Group II: Asymptomatic infection CD4 + T-cell count at normal levels
C. Group III: Persistent generalized lymphadenopathy CD4 + T-cell count 200-500 cells/mm3,
typically asymptomatic although problems such as oral candidiasis, herpes zoster, bacterial
infections and tuberculosis are an increased risk
D. Group IV: Other diseases CD4 + T-cell count <200 cells/mm3, CDC-defined AIDS, often >10
years after being infected
1. Subgroup A: Constitutional Disease: fever, weight loss, and diarrhea
2. Subgroup B: Neurologic disease: dementia, encephalopathy, and myopathy
3. Subgroup C: Secondary infectious diseases
Protozoan parasites:
Pneumocystis carinii (PCP)
Toxoplasma gondii
Cryptosporidium parvum
Fungal:
Candida albicans (thrush)
Cryptococcus neoformans
Aspergillus sp.
Viral:
Cytomegalovirus (CMV)
Herpes Simplex Virus (HSV)
Herpes zoster
Epstein-Barr Virus (EBV)
Bacterial:
Mycobacterium tuberculosis (MTB) / M. avium intracellulare (MAC)
Treponema pallidum (syphilis)
4. Subgroup D: Secondary neoplasms Kaposi’s sarcoma, Non-Hodgkin’s lymphoma, Primary
brain lymphoma
5. Subgroup E: Other conditions
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 53
IV. Treatment
A. Highly active antiretroviral therapy (HAART): antiretroviral regimen capable of suppressing HIV
replication to undetectable levels and sustaining this suppression for many months and perhaps
years in a significant number of people, normally 3 different drugs (protease inhibitor and 2
nucleoside analogues)
B. Prophylaxis
1. Primary: Before patient exhibits symptoms
3
T4 Cell Count <200 cells/mm : Pneumocystis carinii
3
T4 Cell Count <100 cells/mm : Toxoplasma gondii
3
T4 Cell Count <50 cells/mm : MAC, CMV
2. Secondary: After treatment of symptomatic infection, infections are never cured
V. Laboratory Diagnosis
A. Detection of antibodies in patient serum to HIV proteins: Normal window period of 3-4 weeks
(patient has been infected but has not produced detectable level of antibody), six months after
infection antibodies are delectable in 95% of infected individuals
1. NE Legal Aspects of HIV antibody detection
a. Patient consent
b. Results to ordering physician only
c. EIA screen x 2 positive, Western Blot positive: all on same specimen
d. Report to Health Department
2. Enzyme-linked Immunosorbent
Assay (ELISA, EIA)
a. Screening test, 99% sensitive
& specific
False (+): multiparous
women, recently vaccinated
against influenza or hepatitis
B, multiple blood transfusions,
autoimmune disease, other
conditions
False (-): very early or very
late in disease
b. Specimen: patient serum
c. Antigen: HIV grown in cell
culture, the entire cell culture
is killed releasing killed virus
d. Method:
1. Serum added to wells coated with HIV cell culture lysate
2. Incubate / Wash
3. Enzyme-tagged anti-antiglobulin added to test wells
4. Incubate / Wash
5. Add substrate that will create color change with enzyme
6. Stop reaction
7. Read with spectrophotometer: ↑color = ↑absorbance = ↑antibody titer
HIV antigen coated well (Ag) + antibodies in pt serum (Ab) → Ag-Ab complex
e
Ag-Ab complex + AHG with enzyme → Ab-Ag-AHG complex
e
Ab-Ag-AHG complex + Substrate = color development
8. All positive specimens are repeated (i.e., tested in duplicate)
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 54
3. Western Blot
a. Confirmatory test, specificity & sensitivity: >99%
b. Specimen: patient serum
c. Antigen: HIV particles are electrophoreses and transferred to nitrocellulose paper
d. Method:
1. Add patient serum & controls to strips/incubate
2. Wash
3. Add anti-human IgG/enzyme - incubate
4. Wash
5. Add substrate to develop color with enzyme
6. Patient strips are compared to positive and negative control strips
7. Results:
i. Positive test: presence of any two of the following bands: p24, gp41,
gp120/160
ii. Negative test: absence of all bands
iii. Indeterminate test: a single band or other bands not included in definition
of a positive test (nonspecific antibody responses, autoimmune disease,
severe liver disease, malignancies, HIV-2 infection)
Repeat after 6 months, if still indeterminate considered negative
iv. False negative: may lose Ab late in disease, failure to seroconvert
4. IFA Confirmatory test: uses live HIV
cells
B. Detection of HIV antigens
1. EIA / ELISA antigen assays “capture”
HIV antigen, usually p24, from a blood
sample on a solid phase material
Known Ab to HIV + patient serum
with HIV Ag → Ab-Ag complex
Ab-Ag complex + known Ab to HIV
e
with enzyme → Ab-Ag-Ab complex
e
Ab-Ag-Ab complex + Substrate =
color development
2. Early diagnostic aid: screening test in
blood donors to reduce the window periods, infants born to HIV-infected mothers
C. Viral nucleic acid detection: Molecular diagnostic techniques can detect minute quantities of HIV
nucleic acids Know as viral load
Plasma levels of viral RNA are used to:
Monitor progression of the disease after determination of set point
Monitor effectiveness of antiretroviral therapy
Diagnosis of patient with end-stage disease
Early diagnosis of infants born to HIV-infected mothers
D. CD4 (T helper) cell count - close relationship between clinical manifestations of HIV infection and
CD4 cell count (<200 cells/mm3 = AIDS)
E. T4/T8 ratio (T helper/T suppressor cell ratio)
Normal 1-1.5
Suggestive of HIV infected individuals = < 0.5
F. Clinical and laboratory data must be integrated for the evaluation / management of HIV infected
individuals
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 55
Human Immunodeficiency Virus (HIV)
Rotation II Study Questions
Short Answer:
1. State the procedure to follow if a patient has a positive EIA test for HIV antibody.
2. For each of the following categories, list at least two examples of opportunistic infections often seen
in HIV infected patients.
a. Bacterial:
b. Fungal:
c.
Protozoan:
d. Viral:
e. Neoplastic:
3. How is the HIV virus readily killed in the laboratory?
4. According to the Center for Disease Control (CDC), which bands need to be present to consider a
Western Blot test positive for HIV viral infection?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 56
Multiple Choice:
5. The human immunodeficiency virus is a:
a. Coxsackie virus
b. Togavirus
c. Herpes virus
d. Retrovirus
6. The mode of transmission for HIV is:
a. Blood products
b. Congenital
c. Venereal
d. Intravenous drug abuse
e. All of the above
7. As AIDS progresses, the quantity of ____________________ diminishes, and the risk of
opportunistic infection increases.
a. HIV antigen
b. HIV antibody
c. CD4 + T lymphocytes
d. CD8 + T lymphocytes
8. HIV can infect all of the following cells EXCEPT:
a. Helper-inducer subset of T lymphocytes
b. Macrophages
c. Monocytes
d. Polymorphonuclear leukocytes
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 3: Infectious Diseases – HIV
Page 57
CLS419 – Clinical Microbiology II
University of Nebraska Medical Center
Clinical Laboratory Science Program
Part Four:
Autoimmunity and
Autoimmune Diseases
CLS 419 Clinical Microbiology II
Serology
Page 58
AUTOIMMUNITY
Objectives:
After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation for CLS 419, the Clinical Laboratory Science student will be able to:
1. Describe concepts of autoimmunity.
2. Describe factors leading to autoimmunity.
3. Differentiate between organ specific and non-organ specific (multi-system) autoimmunity.
4. Discuss both specific and non-specific responses in autoimmune diseases.
a. Inflammation
b. Hypersensitivity
5. Explain how each of the following tests is used in the diagnosis of autoimmune disorders, including
principle, sensitivity and specificity, and interpretation of results:
a. Anti-nuclear antibody (ANA) detection
b. Anti-DNA antibody detection
6. Define what is meant by an extractable nuclear antigen (ENA), providing three examples.
7. Describe the visual appearance on a Hep-2 cell substrate, mitosis staining reaction and specific
antigen in the nucleus to which the antibody is directed, for each of the following patterns:
a. Homogeneous
b. Centromere
c. Peripheral (rim)
d. Nucleolar
e. Speckled
8. Discuss the significance of the following antibodies, including a disease state they are associated
with:
a. Anti-centromere
e. Anti-RNA
b. Anti-DNA
f. Anti-Smith
c. Anti-ENA
g. Rheumatoid factor
d. Anti-histone
9. Correlate the clinical symptoms, pathophysiology and laboratory findings in the following autoimmune
disorders:
a. Systemic lupus erythematosus (SLE)
b. Rheumatoid arthritis
10. Interpret results of the following tests concerning SLE:
a. ANA
b. Anti-DNA antibody
c. Anti-Sm antibody
d. Anti-histone antibody
11. Identify the affected target tissue (i.e., liver) and the main autoantibody specificity (i.e. AMA) with
each of the following organ specific autoimmune disorders:
a. Hashimoto’s thyroiditis
b. Grave’s disease
c. Pernicious anemia
d. Type I diabetes mellitus
e. Chronic active hepatitis
f. Acute biliary cirrhosis
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 59
Autoimmunity and Autoimmune Disorders
I.
The concept of autoimmunity
A. Autoimmunity represents a state of immunologic self-reactivity, specificity of self-antigens or autoantigens by immunoglobulins or T-cells. There is a breakdown of the immune system’s ability
to discriminate between self and non-self. Autoimmunity is a disruption of the normal tolerant
state the body has to self-components.
B. Classification of autoimmune diseases
Autoimmune disease are grouped based on the type of antibody that is produced; antibodies can
be produced to antigens specific for organs affected by the disease or antibodies can be produce
to non-organ specific constituents such as nucleoproteins or connective tissue.
1. Multi-system - react with nuclear or cytoplasmic antigens
Examples: Systemic lupus erythematosus (SLE)
Scleroderma
Rheumatoid arthritis (RA)
a. Antibodies and lesions are non-organ specific
Rheumatic or connective tissue diseases are non-organ specific autoimmune diseases
producing antibodies to nuclear antigen – ANA.
When these antibodies react with their specific antigen, immune complexes are formed
that may become deposited in various areas of the body. The immune complexes
activate the complement system that results in inflammation of the area surrounding the
complexes.
b. Antigens are accessible at higher concentrations (as compared to organ specific
disorders)
c. Lesions caused by deposition of antigen-antibody (immune) complexes
2. Organ-specific - resulting tissue damage and autoantibodies produced are directed at a
single target organ
Examples: Thyroiditis
Chronic liver diseases (primary biliary cirrhosis, chronic active hepatitis)
Pernicious anemia
Myasthenia gravis
a. Antibodies and lesions are organ specific
b. Antigens only available to lymphoid system in low concentrations
c. Tendency to develop cancer in the organ
C. Factors influencing the development of autoimmunity
1. Genetic factors
2. Age
3. Exogenous factors
a. Viral transformation
b. Drug transformation
c. Newly exposed antigens
II. Multi-system diseases (systemic)
A. Systemic lupus erythematosus (SLE, Lupus)
The prototype of immune complex disease is SLE
1. Disease State
a. Autoimmune systemic disease involving mostly skin, kidneys, joints and serous
membranes
b. Vasculitis (i.e., inflammation of a vessel) usually involving many organ systems
c. Skin lesions usually in the form of a red rash across the nose and upper cheeks maculopapular (butterfly) rash or photosensitivity
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 60
d. Symptoms may include: Fever, weight loss, malaise, arthralgia (joint pain) arthritis,
increased susceptibility to common opportunistic infections, glomerulonephritis, photoinduced skin lesions, anemia, migratory arthritis without joint destruction, myalgia, pleural
and pericardial effusions, seizures, alopecia, hemolytic anemia, purpura, nephritis (50%
have central nervous system involvement), and peripheral neuropathy. The symptom
complex of SLE is inconsistent in duration, severity, and combination of individual
symptoms.
e. SLE occurs more frequently in women than men. Non-white women in early and middle
adulthood have the highest mortality rate. Prior to corticosteroid treatment, average life
expectancy for SLE was two years. Also, only more obvious and advanced cases of SLE
were recognized. With treatment a nearly normal life span is expected.
2. SLE Laboratory Diagnosis
a. ANA’s (antinuclear antibodies) are present
Detection of ANA in patient’s serum by indirect fluorescence utilizing Hep-2 Cells (human
epithelial cells) as a substrate
(See color plates on Blackboard)
Major Patterns
Antibody to:
Characteristics
Homogeneous
DNA, Histones
• Uniform fluorescence of entire nucleus
(diffuse)
• Mitotic cells stain
Peripheral (rim)
DNA
• Fluorescence of entire nucleus w/more
intensity around rim
• Mitotic cells stain
Speckled (fine &
coarse)
Extractable
nuclear antigens
(ENA)
• Numerous small uniform points of
fluorescence scattered throughout the
nucleus
• Mitotic cells do not stain
Nucleolar
Nucleolar RNA
• Intense fluorescence of nucleoli, 2-7 per cell
• Mitotic cells do not stain
Centromere
(discrete speckled)
Centromere
• In mitotic cells, discrete speckles are
clustered only in the chromosome mass,
metaphase & anaphase more defined bar
shapes
b. LE cell: either a normal segmented neutrophil or other phagocyte cell with engulfed
homogeneous and swollen nucleus of either a neutrophil or lymphocyte
In vitro test: expose PMN nucleus, LE factor (an antinuclear antibody) will attach to
nucleus and cause the nucleus to under go changes, it will round up and become
homogeneous, neutrophils will then see the changed nucleus as foreign and will
phagocytize it
c. Detection of autoantibodies of nDNA
(dsDNA) via indirect fluorescence
Substrate - hemoflagellate: Crithidia
lucilliae’s kinetoplast rich in nDNA
d. EIA tests developed for specific antibody
detection
e. Double immunodiffusion used for detection of specific antibodies
f. Sensitivity & specificity of tests for SLE:
Not specific
ANA by IFA Sensitive
nDNA
Sensitive
Specific
LE cell prep Not sensitive Specific
g. Other laboratory findings: Increased serum immunoglobulins Decreased levels of C3 and
C4
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 61
B. Rheumatoid Arthritis (RA)
1. Chronic inflammatory disease primarily affecting the joints and periarticular tissues due to the
formation of immune complexes. It is a debilitating disease affecting women two or three
times more frequently than men. It can occur at all ages but most commonly in the fourth
decade.
2. Symptoms may include: fatigue, weakness, anorexia, weight loss, and malaise with fever and
joint pain. Stiffness often develops after periods of inactivity and eventual muscle atrophy
near affected joints.
3. The inflammatory joint changes, which most often affect the small joints, may result in loss of
function or permanent deformity
4. Rheumatoid Factor: several abnormal proteins
circulate in the blood of patients with RA. They
are collectively known as “rheumatoid factor.”
They are a group of immunoglobulins (IgG, IgM
and rarely IgA) that interact specifically with the
Fc portion of IgG.
Rheumatoid Factor can occur in non-rheumatoid
individuals with chronic infections (i.e., SLE,
infectious hepatitis, chronic hepatic disease,
syphilis)
5. Screening test for rheumatoid factor
a. Latex agglutination: latex particles coated with IgG molecules
b. If rheumatoid factor is present it attaches to the Fc portion of IgG and agglutinates latex
particles
C. Scleroderma (SCL) or Progressive Systemic Sclerosis (PSS)
1. PSS is a generalized disorder of the connective tissue characterized by diffuse fibrosis
involving the skin and several internal organs (lung, heart, kidney and GI tract). This collagen
vascular disease affects women more often than men.
2. Degenerative changes of the skin (scleroderma)
3. Vascular abnormalities – Reynaud’s phenomenon: pain in the extremities when exposed to
cold temperatures
4. CREST syndrome: is a milder form of scleroderma most often limited to the skin (often face
and fingers only)
Calicinosis: abnormal calcium deposits in tissue
Raynaud’s phenomenon
Esophageal dysmotility: difficulty in swallowing
Sclerodactyly: scleroderma of fingers & toes
Telangiectasia: dilation of a group of small blood vessels
5. Laboratory findings: Positive ANA with speckled or nucleolar pattern With CREST see
centromere pattern
D. Mixed Connective Tissue Disease (MCTD)
1. Combined clinical features of SLE, Scleroderma and RA with no kidney involvement
2. Laboratory findings: Positive ANA with speckled pattern Can have RF
Hypergammaglobulinemia AntiDNA is absent
E. Sjögren’s Syndrome (SS)
1. SS is a chronic inflammatory rheumatic disease and often occurs secondary to RA,
scleroderma, SLE or polymyositis (inflammation of numerous muscles at once)
2. Symptoms include dry eyes (lack of tears) and dry mouth with scanty, sticky saliva. The
disease can occur at any age but is most often occurring in women between 30-60 years.
Approximately 50% of patients have intermittent salivary gland enlargement, and
approximately 50% of patients will also have rheumatoid arthritis.
3. Laboratory findings: Positive ANA with speckled pattern RA factor Hypergammaglobulinemia
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 62
F. Review of ANA patterns by IFA with disease states
(See color plates posted on Blackboard)
Major Patterns
Antibody to:
Characteristics
Homogeneous
DNA, Histones
• Uniform fluorescence of entire nucleus
(diffuse)
• Mitotic cells stain
Disease
SLE, MCTD
Peripheral (rim)
DNA
• Fluorescence of entire nucleus w/more
intensity around rim
• Mitotic cells stain
SLE
Speckled (fine &
coarse)
Extractable
nuclear antigens
(ENA)
• Numerous small uniform points of
fluorescence scattered throughout the
nucleus
• Mitotic cells don’t stain
SLE, RA,
MCTD,
PSS, SCL
Nucleolar
Nucleolar RNA
• Intense fluorescence of nucleoli, 2-7
per cell
• Mitotic cells don’t stain
PSS, SLE
Centromere
(discrete
speckled)
Centromere
• In mitotic cells, discrete speckles are
clustered only in the chromosome mass,
metaphase & anaphase more defined
bar shapes
CREST,
SLE
III. Organ/Tissue Specific Diseases
A. Chronic Liver Disease
1. Primary biliary cirrhosis:
a. Unknown etiology, inflammatory reaction of bile ducts, jaundice, putitis, malabsorption,
may be self-limiting or ducts become more occluded leading to cirrhosis and end stage
lover disease
b. Serology: anti-mitochondrial antibody (AMA)
2. Chronic active hepatitis:
a. Inflammation of liver with dysfunction
b. Serology: anti-smooth muscle antibody (ASMA)
B. Pernicious anemia:
1. Inflammation of gastric mucosa with inability to secrete HCL, intrinsic factor and pepsin
followed by the development of macrocytic anemia
2. Serology: anti-parietal cell and anti-intrinsic factor
C. Thyroid Diseases
1. Hashimoto’s Thyroiditis (Autoimmune Thyroiditis):
a. Enlargement of thyroid, hypothyroid
b. Serology: Anti-thyroglobulin antibody Anti-microsomal antibody
2. Grave’s Disease:
a. Hyperthyroidism, 50% have long-acting thyroid stimulator (LATS), 90% have thyroidstimulating immunoglobulins (TSI)
b. Serology: Anti-TSH receptors
D. Other Examples of Organ or Tissue Specific
1. Diabetes (Juvenile): anti-pancreatic cell
2. Addison’s disease: anti-adrenal cortex
3. Myasthenia gravis: anti-acetylcholine receptor
4. Multiple sclerosis (MS): anti-myelin sheath
5. Autoimmune hemolytic anemia: anti-red cell antigens
6. Goodpasture’s disease: anti-glomerular basement membrane
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 63
Correlation of Specific Antinuclear Antibodies
1. Nuclear autoantibodies are common to many connective tissue diseases including SLE, RA, SS, SCL
or PSS, and MCTD. Since the nuclear autoantibodies are directed against specific components of the
nucleus, characteristic fluorescent patterns are demonstrated. However, a given autoantibody may be
associated to more than one fluorescent pattern (i.e., homogeneous versus peripheral) and a given
pattern may have several corresponding autoantibodies (i.e., speckled: SM, RNP, Scl-70, SSB, etc.).
Consequently, a lucrative correlation exists between fluorescent patterns and specific connective
tissue diseases.
NOTE: Patient sera containing more than one antinuclear antibody at different strengths may result in
fluorescent patterns changing with progressive dilutions.
2. Often specific identification of nuclear autoantibodies or groups of autoantibodies correlates better to
one or more connective tissue disease than the fluorescent pattern. Occasionally quantitation of
these autoantibodies has been useful in differentiation among connective tissue diseases. Certain
nuclear autoantibodies are highly specific and tissue disease. Certain nuclear autoantibodies are
highly specific and may e considered markers for a given connective tissue disease.
3. When 3 or 4 different ANA patterns are observed simultaneously the probability of SLE is
approximately 90%.
4. The normal population usually has negative results of autoantibodies. However, apparently healthy
people over 50 years may have positive results without association to disease.
5. Characteristics associated with specific antinuclear antibodies:
a. Deoxyribonucleic acid (DNA)
At least 3 major classes of antibodies to DNA have been identified:
Antibodies to double-stranded DNA (dsDNA)
Antibodies to single-stranded DNA (ssDNA)
Antibodies with specificity for both dsDNA and ssDNA
Antibodies to dsDNA and antibodies with specificity for ssDNA and dsDNA are often referred to
as antibodies to native DNA (nDNA).
Antibodies to only dsDNA are found in SLE but are relatively rare. These antibodies produce a
rim and/or homogeneous IFA pattern.
Antibodies to ssDNA are found most frequently in SLE but can also be found in other rheumatic
and non-rheumatic diseases. ssDNA antibodies are not detected by routine IFA.
The DNA antibodies with reactivity for ssDNA and dsDNA are the most frequent DNA antibodies
in patients with SLE and produce a rim and/or homogeneous pattern. These antibodies can be
found in other rheumatic diseases in low titer.
High levels of nDNA antibodies have a positive correlation with SLE and nephritis due to the
presence of DNA-anti-nDNA immune complexes causing inflammation of the kidney. (Antibodies
to dsDNA fix complement and form insoluble complexes that are deposited in the basement
membrane of the glomerulus, resulting in glomerulonephritis and proteinuria.) Immune complexes
may also lodge n the skin and vascular system.
b. Deoxyribonucleoprotein (DNP)
Antibodies to deoxyribonucleoprotein (DNP) have been called the LE cell antibodies, as they are
responsible for the LE cell phenomenon that is positive in 60-70% of SLE cases. DNP antibodies
are relatively specific for SLE and produce a rim and/or homogeneous pattern with IFA testing.
Antibodies to DNP can be measured by RIA, EIA, immunodiffusion, CIE or passive agglutination.
A latex agglutination test for DNP antibodies has been reported to lack sensitivity and is not
recommended for screening.
c. Histone
Histone antibodies also produce a homogeneous immunofluorescent pattern. They are found in
patents with drug induced lupus usually to the exclusion of other types of ANA characteristically
found in SLE. Histone antibodies can also be detected in SLE patients along with other antibodies
found in SLE. Histone antibodies can be identified with an immunofluorescent extraction
procedure or more recently developed RIA and ELISA techniques.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 64
d. Extractable Nuclear Antigens (ENA)
Extractable nuclear antigens (ENA) are non-histone antigens that are extractable form calf or
rabbit thymus. A number of ENA antibodies have been identified.
i. Smith (Sm)
The Sm antibodies are considered highly specific marker antibodies for SLE and are found in
25-30% of these patients. Sm antibodies are rarely found in other connective tissue diseases.
Sm antibody produces a speckled fluorescent pattern.
ii. Ribonucleoprotein (RNP)
The RNP antigen is closely associated with the Sm antigen. Anti-RNP can be found in a
variety of rheumatic diseases such as SLE, RA, SS, PSS, and MCTD. Patients with MCTD
have high titers of RNP antibodies to the exclusion of other types of ANA.
iii. Sjögren’s Syndrome
SSA and SSB antibodies are identical to the previously described Ro and La antibodies
respectively.
SSA antibodies are found in 70% of patients with Sjögren’s Syndrome and 40% of SLE
patients. SSB antibodies, although found in SLE, are primarily considered a marker antibody
for Sjögren’s Syndrome and are found in 48% of these patients. Recently there has been a
great deal of interest in the association of SSA antibodies, neonatal lupus and congenital
heart block. The SSA antibodies are very significant to detect especially in pregnant women.
SSB antibodies produce a speckled fluorescent pattern. SSA antibodies are not detected in
tissue substrates due to the low antigen concentration. Human cell lines such as the Hep-2
cell contain enough antigens to detect the SSA antibody when the substrate has been
properly fixed. The immunofluorescent pattern is a speckled reaction.
e. Scleroderma-70 (Scl-70)
Scl-70 antibodies are very specific for PSS patients but are found only in 2030% of these
patients. Scl-70 antibodies produce a finely speckled staining with or without nucleolar staining.
The Scl-70 antigen has been found to be chromosome associated and therefore will show a
positive chromosome reaction with Hep-2 cells.
f. Nucleolar
Nucleolar antibodies can be detected in up to 90% of PSS patients, usually in high titers. These
antibodies can be found in other rheumatic diseases such as SLE but generally in low titers.
Nucleolar antibodies produce the characteristic staining of nucleoli that may appear homogenous
or speckled. Tissue substrates contain 1-3 nucleoli, while tissue culture cells such as Hep-2 cells
may contain 2-7 irregular shaped nucleoli.
g. Centromere
Centromere antibody is found in a high percentage of patients with CREST syndrome, a less
severe variant of PSS. Centromere antibody can be found in other rheumatic diseases and may
predate a full-blown CREST syndrome by as early as 2 years.
Centromere antibody produces a discrete immunofluorescent speckled pattern on tissue or Hep2, but can be positively identified on a Hep-2 cells by observing the chromosome staining of the
mitotic cells where the speckles are clustered with the chromosomes.
5. Characteristics associated with other autoantibodies:
a. Mitochondrial (AMA)
A high AMA titer supports the diagnosis of primary biliary cirrhosis. Low titers of AMA may be
detected in other liver disorders that include chronic active hepatitis and cryptogenic cirrhosis.
b. Smooth Muscle (ASMA)
ASMA is present in high titers in the serum of 70% of patients with chronic active hepatitis. In
addition, 50% of these patients are positive for ANA, while 25% demonstrate low AMA titers. Low
ASMA titers may be present in viral infections, malignancies, and good health. ASMA usually
does not appear in SLE.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 65
c.
Parietal Cell (APCA)
APCA occurs in the serum of 90% of patients with pernicious anemia. With other clinical and
laboratory data, a positive APCA result helps to distinguish autoimmune pernicious anemia from
other megaloblastic anemias. Other disorders that may have APCA are gastric ulcers (33%),
atropic gastritis (up to 60%), thyroid diseases (33%), diabetes mellitus (12%), and iron deficiency
anemia (24%). Although detected in less than 2% of the normal population under 20 years of age,
the incidence of APCA increase in women over the age of 40 and may be present in up to 16% of
the normal population over 60 years of age.
Profiles
Screening for antinuclear antibodies aids the physician in evaluating systemic rheumatic disease.
Specific identification of antibodies aids in diagnosis as certain disease produce specific antibody
profiles.
ANA Profiles:
Antibodies to:
Disease
nRNP
nDNA
Histone
Sm
SSB
SSA
+
+*
+*
+*
+*
+
+
+
-
+
-
+
+
+
+
+
+
+
SLE
+
MCTD
+
Scleroderma
+
DM/PM
Sjögren’s
+
RA
Drug induced lupus
+*
-
Scl70
+
-
Centromere
+
-
Nucleolus
+
+
-
DM / PM
Ag
+
+
-
* Low titers
ƒ The presence of multiple antibodies suggests SLE. High tittered nDNA antibodies are also
characteristic of SLE and anti-Sm is a marker for SLE.
ƒ Patients with MCTD usually have high titers of anti-RNP in the absence of other types of ANA.
ƒ Drug induced lupus presents with histone antibodies without other ANA.
ƒ In Sjögren’s Syndrome both SSA and SSB are likely to be present. These antibodies can also be
found in SLE.
ƒ Scl-70 and nucleolar antibodies are associated with PSS, and centromere antibodies are found in
CREST syndrome.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 66
DNA Testing
Principle:
The indirect fluorescent immunoassay principle has been discussed in earlier sections. Initially, serum is
applied to the Crithidia lucilliae substrate. If there are anti-nDNA antibodies present they will attach to the
kinetoplast forming an Ag-Ab complex. After removing the excess serum from the slide, a fluorescein
tagged anti-human gamma globulin is applied that attaches to any Ag-Ab complexes already present.
Once this material is removed, the slide is viewed under a fluorescent microscope. In a positive reaction,
the kinetoplast will appear apple green while the negative will appear dull or dark. Since patients with SLE
can have antibodies other than nDNA, the value of the test is in a positive result. While a positive is highly
indicative of SLE, a negative result would not rule out SLE.
Clinical Value:
The anti-nDNA assay is an aid in the diagnosis and treatment of SLE by detecting and titering anti-double
stranded (native) deoxyribonucleic acid (nDNA) antibodies in human serum. Many auto-antibodies have
been associated with SLE. Some of these also share associations with other diseases as well. The group
of DNA antibodies can be divided into three classes:
a. nDNA antibodies
b. ssDNA antibodies
c. Antibodies that react with both of the above classes
Of these, those directed against nDNA antibodies have gained most importance because of their
diagnostic and therapeutic value. It is generally believed that high titers of nDNA antibodies occur only in
SLE. The others can be found in SLE or other diseases as well. The titer of nDNA antibodies may also
respond to therapy by decreasing with successful treatment and increasing in an acute disease state.
The anti-nDNA test is an indirect immunofluorescent test that utilizes the protozoan, Crithidia lucilliae, as
the substrate. This method was chosen over others because the Crithidia possess a giant mitochondria
called the kinetoplast that is composed of pure nDNA. Unlike some of the other methods that could easily
become contaminated with ssDNA the composition of the Crithidia is consistent.
Results:
1. Positive
In a positive reaction, the kinetoplast fluoresces greater than the negative as a 1:10 dilution. The
following are all considered positive:
a. Only the kinetoplast fluoresces
b. The kinetoplast and nucleus fluoresce
c. The kinetoplast and basal body fluoresce
d. The kinetoplast, basal body and nucleus
fluoresce
2. Negative
In a negative reaction, the kinetoplast does not
fluoresce at a 1:10 dilution. The following are all
considered negative:
a. No fluorescence
b. The nucleus fluoresces
c. The basal body fluoresces
d. The basal body and nucleus fluoresce
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 67
Autoimmunity and Autoimmune Disorders
Rotation II Study Questions
Matching:
1. Match the following autoantibodies with the most likely disease state indicated by their presence
when found alone:
____ Anti-mitochondrial
____ Anti-parietal cell
____ Anti-smooth muscle
____ Anti-centromere
____ Anti-DNA
____ Anti-dsDNA
____ Anti-histone
____ Anti-Sm
____ Anti-thyroglobulin
a. Systemic lupus erythematosus (SLE)
b. Drug induced SLE
c. CREST syndrome
d. Chronic active hepatitis
e. Pernicious anemia
f. Primary biliary cirrhosis
g. Hashimoto's thyroiditis
2. Match the following fluorescent patterns on Hep-2 slides with the antibody being expressed:
Fluorescent pattern on Hep-2 slides
____ Speckled
____ Homogeneous
____ Nucleolar
____ Centromere
Antibody being expressed
a. Anti-DNA
b. Anti-centromere
c. Anti-histones
d. ENA’s (Anti-Scl-70, Anti-SSA, Anti-SSB)
e. Anti-RNA
Multiple Choice:
Choose the single best answer.
3. Which of the following is NOT a common feature of autoimmune diseases?
a. Elevated serum immunoglobulins
b. Decreased complement levels
c. High incidence in females
d. Lesions due to deposit of immune complexes
e. Disease will not respond to steroid therapy
4. Some mechanisms responsible for the induction of autoimmunity include:
a. Release of normally sequestered cell components
b. Loss of T-suppressor cells
c. Altered tissue components
d. All of the above
e. B and C only
5. A patient's ANA result is as follows:
Pattern: Homogeneous
Titer: 1:640
Which of the following would most likely be done next?
a. Anti-DNA testing
b. Report as normal
c. Anti-smooth muscle testing
d. Anti-extractable nuclear antigen testing
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 68
6. A 78-year-old male patient admitted to the hospital with angina had the following ANA results:
Pattern: Homogeneous
Titer: 1:40
Which of the following is most likely the cause?
a. Systemic lupus erythematosus
b. Syphilis
c. Age-related false positive
d. Anti-RNA
7. All of the following characteristics are common to organ-specific and non-organ-specific disorders
EXCEPT:
a. Autoantibody tests are of diagnostic value
b. Antibodies may appear in each of the main immunoglobulin classes
c. Antigens are available to lymphoid system in low concentrations
d. Circulatory autoantibodies react with normal body constituents
8. Antibody expression in the development of autoimmunity is regulated by all of the following factors
EXCEPT:
a. Genetic predisposition
b. Increasing age
c. Environmental factors (e.g., UV radiation)
d. Active infectious disease
9. The mechanism responsible for autoimmune disease is
a. Circulating immune complexes
b. Antigen excess
c. Antibody excess
d. Antigen deficiency
10. One of the mechanisms believed to induce self-tolerance is
a. Induction of responsiveness in immunocompetent cells
b. Elimination of clone programmed to react with antigen
c. Decreased suppressor cell activity
d. Stimulation of clones of immunocompetent cells
Short answer:
11. Indirect fluorescent antibody testing detecting AMA, ASMA and APCA patterns utilizes
______________________________ substrate.
12. The presence of multiple autoantibodies typically suggests ______________________________.
13. Briefly summarize the indirect fluorescent procedure as used for autoantibody testing.
14. DNA testing is best determined on ______________________________ substrate. If anti-dsDNA
antibodies are present, they will attach to the ______________________________ organelle. A
positive DNA test can help diagnose ______________________________.
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 69
15. Describe the staining pattern as seen in each of the following: (including appearance of both nucleus
and chromosomes)
Nucleus
Chromosomes
a. Homogenous
b. Nucleolar
c.
Rim
d. Speckled
16. Immune injury in rheumatoid arthritis is due to the formation of soluble circulating antigen-antibody
complexes. Give the nature of the soluble circulating complex.
17. What autoantibody is present in pernicious anemia?
18. What pattern of fluorescence is produced by anti-DNA, -DNP, -Histone in the IFA test?
19. What disorders would be suggested by the rim or homogeneous pattern of fluorescence on IFA and
what follow-up tests would be suggested?
20. What test would be used to determine if a speckled pattern were due to antibody against Scl that is
highly diagnostic of scleroderma?
CLS 419 Clinical Microbiology II
Rotation 2
Serology
Module 4: Autoimmunity and Autoimmune Diseases
Page 70