Influenza Serologic Diagnosis and Epidemiology

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Influenza Serologic Diagnosis and Epidemiology Powered By Docstoc
					                                      Influenza:
                                      Serologic Diagnosis
                                      and Epidemiology
                                      Marvin A. Cuchens, PhD
                                          Department of Microbiology
                                          University of Mississippi Medical Center
                                          Jackson, MS
                                      Bradley S. Bender, MD
                                          Department of Medicine
                                          University of Florida College of Medicine
                                      Steven Taffet, PhD
                                          Department of Microbiology and Immunology
                                          SUNY Upstate Medical University
                                          Syracuse, NY

                                      Originally Developed by:
                                      Parker A. Small, Jr., MD
                                      R. G. Weber, PhD
                                          Department of Immunology and Medical Microbiology
                                          College of Medicine, University of Florida
                                      Susan M. Johnson, PhD
                                          College of Pharmacy. University of Florida
                                          Gainesville, Florida

                                      Modified by:
                                      Jean-Michel Goust, M.D.
                                      Gabriel Virella, M.D., Ph.D.
                                          Medical University of South Carolina, Charleston, SC




The reason the POPS system works so well is that they have been revised many times based on feedback from
students and faculty.
Please send suggestions and/or comments to: mcuchens@microbio.umsmed.edu
         Note to Instructors
         This workbook is divided into five sections:
         1. Introduction to the POPS System, introduction to and objectives of the clinical
             simulation, and a pretest
         2. Four booklets with pretest answers and the clinical problem(s)
         3. Group question and answer sheets
         4. Posttest
         5. Posttest answers
Each student should receive a copy of section 1 to study and answer questions before the group
problem-solving session. If you wish, section 2 also may be distributed for the students to review
prior to the group session.
                                      A Patient-Oriented Problem-Solving (POPS) System
                                      System supported by the American Medical Association Education and Research Foundation

                                                                                                                               1
Influenza
Introduction to the Patient-Oriented
Problem-Solving (POPS) System
This is a Patient-Oriented Problem-Solving activity. The purposes are:

1.   To help you learn how to apply your basic science knowledge to the solution of clinical problems

2.   To help you learn how to better use sources (ie, textbooks and peers) that will be available to you
     throughout your career

3.   To help you work with your fellow students and thus
     a. increase your ability to evaluate your colleagues’ opinions, thought processes, and diagnoses
     b. increase communications skills
     c. get to know your classmates better

This activity consists of four phases. First, you will review the attached set of objectives, do background
reading on the topics to be covered, and complete the pretest on your own. In the second phase, you will
join three other students and review the pretest answers in an “open-book” discussion. In the third phase,
the group will solve patient-oriented problems. Information exchange and group interaction are keys to the
success of this phase. This process will allow you to teach your fellow students and, at the same time, learn
from them. Finally, you will take a posttest, individually, which will enable you to assess your progress.




                                                                                                       2
Influenza
Introduction
Influenza is an epidemic disease that occurs yearly and causes much suffering and loss of life. Moreover,
at less frequent intervals (eg 1918, 1957, 1968 and 1977), influenza causes world pandemics; the one in
1918 caused 20 million deaths. This Patient-Oriented Problem-Solving activity deals with:
  • The laboratory procedures used to diagnose the disease (the basic concepts are also applicable to the
     diagnosis of many other diseases).
  • The measures that can be taken to prevent the disease in different populations.
  • The mechanism of action of the pharmacological agents that have activity against the influenza virus

Influenza virus and certain other viruses are able to agglutinate red blood cells (RBCs). This is called
hemagglutination (HA). However, in the presence of specific antibody to the virus, HA is blocked. This
phenomenon provides the basis for an assay used to detect and quantitate specific antiviral antibodies in
serum. This test is known as the hemagglutination inhibition (HI) assay.

In this activity, you will be asked to analyze HI data from several patients and to discuss other diagnostic
alternatives and preventive measures against influenza. Upon completion of the group session, you should
be able to:
1) determine, based upon HI data, whether a patient was
   a) infected with the pathogen during the period of acute illness or
   b) infected or immunized prior to that time with an antigenically identical or similar pathogen.
In order to accomplish these objectives, you must be able to:
2) schematically draw the in vitro interactions of virus, RBCs, and antibody in HA and HI assays.
3) interpret an HI assay, determine the HI titer, and defend or dispute the validity of the data based on
   the controls.
4) compare acute and convalescent serum titers to determine whether there has been a significant
   increase in antibody.
5) discuss other diagnostic procedures that can be used and when are they indicated
6) list the preventive measures that are available for influenza, discuss their advantages and limitations.
7) summarize the mechanisms of action of the different ant-viral agents that have activity against the
   influenza virus
In addition to learning these facts and concepts, it is important that you get to know your classmates better
and become more skilled at teaching and learning from each other. Much of your learning during your
clinical training and throughout your professional career will result from interactions with peers, house staff,
and practicing physicians. Improving your ability to be patient with, considerate of, and helpful to your
colleagues is easier now (when the “pressure” comes from a simulated clinical problem) than it will be when
the well-being of an actual patient is at stake. Good habits that you develop now hopefully will continue in
the future.


When you have become familiar with the objectives, complete the pretest on the next page.

                                                                                                          3
Influenza
Pretest
Instructions: Please mark your answers to the following questions on this exam to facilitate later
discussion and review. If your instructor has provided a separate answer form, please be sure to fill in the
identification section; then answer the questions both on the form and on this exam.

Choose the one correct or most appropriate answer. If you do not know an answer, leave it blank. Do not
guess. Health professionals who think they know something, but don't, can do real harm. Those who know
they don't know something can get help.

Don’t be upset if you don't know all the answers. The purpose of the pretest and objectives is to alert you to
important concepts. The posttest will be similar to the pretest.

1.     What is the minimal number of red blood cell (RBC) binding sites required on a virus for the virus to
       agglutinate RBCs?
       (A)    0
       (B)    1
       (C)    2
       (D)    10
       (E)    >10

Questions 2 and 4 relate to the following hemagglutination (HA) assay:

A 1.0 ml sample of influenza virus suspension was added to test tube #1, and 0.5 ml of saline was added to
tubes #2 through #9. A 0.5 ml sample of the virus suspension was removed from tube #1 and added to tube
#2. After mixing, 0.5 ml was removed from tube #2 and added to tube #3. This proceeded until all the
dilutions were made and 0.5 ml from tube #9 was discarded. Tube #10 contained only 0.5 ml of saline.
Chicken RBCs were then added to all the tubes, and the following pattern was observed. (Each circle
represents one test tube viewed from above.)




2.     Which tubes have agglutinated cells in them?
       (A)   1 through 7
       (B)   8 through 10
       (C)   8 and 9
       (D)   10
       (E)   None of the above

3.     What is the dilution of virus suspension in tube #5 (ignoring the volume of RBCs added)?
       (A)    Undiluted
       (B)    1:8
       (C)    l:16
       (D)    1:32
       (E)    None of the above

                                                                                                        4
Influenza

4.     Like most infectious diseases, the diagnosis of influenza is suspected on clinical grounds, and can be
       confirmed by all the following EXCEPT:
       (A)    Isolation of the pathogen
       (B)    Antigen detection
       (C)    IgM titers
       (D)    Rise in IgG titers (acute and convalescent)
       (E)    Elevated complement levels

5.     In October, Mr. Smith, a 66-year old man, comes to your office for his annual physical examination.
        He received an influenza vaccine last fall and wants to know if he needs another one this year. You
       recommend influenza vaccination (an inactivated virus vaccine) to him for all of the following
       reasons EXCEPT:
       (A)    He is over age 65-years, and older persons suffer excessively from influenza.
       (B)    Influenza vaccine is an inactivated vaccine; antibody induced by inactivated vaccines tend
              not to last as long as that induced by live virus infection
       (C)    The influenza virus undergoes periodic antigenic changes; serum antibody is very
              specific, so a new vaccination is needed to induce specific antibody to the current circulating
              strain.
       (D)    Multiple influenza vaccinations induce anti-influenza cellular immunity, which is deficient
              in older persons.

The following hemagglutination inhibition (HI) assay pattern relates to questions 6 through 8.
Circles 1 through 7 represent test tubes to which have been added increasing dilutions of a serum
containing antibody to influenza virus, followed by constant amounts of influenza virus, and finally
RBCs. The controls contain RBCs plus saline (8), RBCs plus serum (9), or RBCs plus virus (10).




6.     Which of the test tubes demonstrate HI?
       (A)   1,2,3,4,8,9
       (B)   5,6,7,10
       (C)   8,9
       (D)   5,6,7
       (E)   1,2,3,4




                                                                                                       5
Influenza
7.    Which of the drawings (A to E below) schematically represents the cellular and molecular events
      taking place in tube #2?



             RBC                    Antibody to virus             Virus



      (A)                           (B)                           (C)




      (D)                           (E)




8.    What is the titer of the serum assayed in the HI test?
      (A)    Less than 10 (<10)
      (B)    80
      (C)    160
      (D)    Greater than or equal to 640 (≥640)
      (E)    Cannot be determined from the data

9.    Which one of the following anti-viral drugs can be used to prevent viral influenza?
      (A)   Acyclovir
      (B)   Gancyclovir
      (C)   Oseltamivir
      (D)   Ribavirin
      (E)   Zydovudine

10.   The antiviral effects of amatadine and rimantidine are related to their ability to:
      (A)    Block the viral RNA-dependent RNA polymerase
      (B)    Block viral protein synthesis
      (C)    Inactivate the cellular DNA-dependent DNA polymerases
      (D)    Inhibit the release of viral nucleocapsids into the cytoplasm of non-infected cells
      (E)    Prevent the splicing of host nascent RNA chains that serve as primers for viral mRNA
             synthesis

11.   The influenza vaccine is best described as a(n):
      (A)    attenuated vaccine
      (B)    component vaccine
      (C)    conjugate vaccine
      (D)    inactivated vaccine
      (E)    recombinannt vaccine

                                                                                                6
 Influenza

 Questions 12 and 13 pertain to the following graph:


        1000         Illness




HI Titer 100




         <10
                          1            2          3              4               1 Year    5 Years
                                       Time (weeks)
         Influenza
         Infection


 12.   Determine from the graph how long it takes to obtain an antibody increase after the onset of an
       influenza infection. (The ILLNESS box represents the duration of clinical disease.)
       (A)    1 to 2 days
       (B)    3 to 6 days
       (C)    1 to 2 weeks
       (D)    3 to 4 weeks
       (E)    >1 month

 13.   Determine from the graph how long detectable antibody lasts.
       (A)   Less than 1 month
       (B)   1 to 11 months
       (C)   1 year
       (D)   5 years
       (E)   Greater than 5 years




                                                                                                 7
Influenza

In answering questions 14 and 15, refer to the HI test below.

Answer true by selecting a.
Answer false by selecting b.
Answer by selecting c if it is impossible to determine whether the statement is true or false from the data
given.
Leave the answer blank if you aren't sure.

Joe Brown had the following HI test results for influenza B antibody. (Joe has never had "flu shots" or other
influenza immunizations.)




14.    Joe had an acute influenza B infection at the time of the test.       a       b       c

15.    Joe had antibody to influenza B in his serum.                         a       b       c




When you have completed the pretest, consult your study materials. Try to identify the correct answers and
understand the concepts that make them correct. The list of objectives may be used as a guideline for your
studies. When your group meets, you will have the responsibility of explaining some of the correct pretest
answers to your groupmates. Please bring your textbook and pretest to the group meeting.




                                                                                                       8
                          Influenza:
                          Serologic Diagnosis
                          and Epidemiology
                          Marvin A. Cuchens, PhD
                              Department of Microbiology
                              University of Mississippi Medical Center
                              Jackson, MS
                          Bradley S. Bender, MD
                              Department of Medicine
                              University of Florida College of Medicine
                          Steven Taffet, PhD
                              Department of Microbiology and Immunology
                              SUNY Upstate Medical University
                              Syracuse, NY

                          Originally Developed by:
                          Parker A. Small, Jr., MD
                          R. G. Weber, PhD
                              Department of Immunology and Medical Microbiology
                              College of Medicine, University of Florida
                          Susan M. Johnson, PhD
                              College of Pharmacy. University of Florida
                              Gainesville, Florida

                          Modified by:
                          Jean-Michel Goust, M.D.
                          Gabriel Virella, M.D., Ph.D.
                              Medical University of South Carolina, Charleston, SC


                               BOOK A
Note to Students
      The fundamental purpose of all activities in health-care professions is to help other people. Like
all behavior, helping behavior becomes more effective and natural with practice. This workbook
enables you to practice by helping your fellow students to learn basic science. Your skill at helping
your fellow students should relate to your ability to help your patients in the future.
      This is a Patient-Oriented Problem-Learning (“POPS”) workbook designed for four students.
Before beginning this session, you should have (a) studied the objectives designed to prepare you for
it, (b) taken the pretest, and (c) reviewed the topics listed at the end of the pretest. Now, each of you
should take one of the four color-coded booklets and follow the directions in it. If your group has
only three students, one of you should take two booklets. Leave the remainder of the workbook
intact until you are given further instructions.
      For additional information, SEE:
http://www.medinfo.ufl.edu/cme/flu/flu.html
http://www.cdc.gov/ncidod/diseases/flu/fluvirus.htm

                          A Patient-Oriented Problem-Solving (POPS) System
                          System supported by the American Medical Association Education and Research Foundation

                                                                                                                   9
Influenza
Introduction to the Patient-Oriented
Problem-Solving (POPS) System
This is a Patient-Oriented Problem-Solving activity. The purposes are:

1.   To help you learn how to apply your basic science knowledge to the solution of clinical problems

2.   To help you learn how to better use sources (ie, textbooks and peers) that will be available to you
     throughout your career

3.   To help you work with your fellow students and thus
     a. increase your ability to evaluate your colleagues’ opinions, thought processes, and diagnoses
     b. increase communications skills
     c. get to know your classmates better

This activity consists of four phases. First, you will review the attached set of objectives, do background
reading on the topics to be covered, and complete the pretest on your own. In the second phase, you will
join three other students and review the pretest answers in an “open-book” discussion. In the third phase,
the group will solve patient-oriented problems. Information exchange and group interaction are keys to the
success of this phase. This process will allow you to teach your fellow students and, at the same time, learn
from them. Finally, you will take a posttest, individually, which will enable you to assess your progress.




                                                                                                      10
Influenza
Introduction
Influenza is an epidemic disease that occurs yearly and causes much suffering and loss of life. Moreover,
at less frequent intervals (eg 1918, 1957, 1968 and 1977), influenza causes world pandemics; the one in
1918 caused 20 million deaths. This Patient-Oriented Problem-Solving activity deals with:
  • The laboratory procedures used to diagnose the disease (the basic concepts are also applicable to the
     diagnosis of many other diseases).
  • The measures that can be taken to prevent the disease in different populations.
  • The mechanism of action of the pharmacological agents that have activity against the influenza virus

Influenza virus and certain other viruses are able to agglutinate red blood cells (RBCs). This is called
hemagglutination (HA). However, in the presence of specific antibody to the virus, HA is blocked. This
phenomenon provides the basis for an assay used to detect and quantitate specific antiviral antibodies in
serum. This test is known as the hemagglutination inhibition (HI) assay.

In this activity, you will be asked to analyze HI data from several patients and to discuss other diagnostic
alternatives and preventive measures against influenza. Upon completion of the group session, you should
be able to:
1) determine, based upon HI data, whether a patient was
   a) infected with the pathogen during the period of acute illness or
   b) infected or immunized prior to that time with an antigenically identical or similar pathogen.
In order to accomplish these objectives, you must be able to:
2) schematically draw the in vitro interactions of virus, RBCs, and antibody in HA and HI assays.
3) interpret an HI assay, determine the HI titer, and defend or dispute the validity of the data based on
   the controls.
4) compare acute and convalescent serum titers to determine whether there has been a significant
   increase in antibody.
5) discuss other diagnostic procedures that can be used and when are they indicated
6) list the preventive measures that are available for influenza, discuss their advantages and limitations.
7) summarize the mechanisms of action of the different ant-viral agents that have activity against the
   influenza virus
In addition to learning these facts and concepts, it is important that you get to know your classmates better
and become more skilled at teaching and learning from each other. Much of your learning during your
clinical training and throughout your professional career will result from interactions with peers, house staff,
and practicing physicians. Improving your ability to be patient with, considerate of, and helpful to your
colleagues is easier now (when the “pressure” comes from a simulated clinical problem) than it will be when
the well-being of an actual patient is at stake. Good habits that you develop now hopefully will continue in
the future.


When you have become familiar with the objectives, complete the pretest on the next page.

                                                                                                         11
Pretest Correct Answers
You have the answers to some of the 18 pretest questions, and other members of your group have the
remainder. This arrangement is designed to encourage all members of your group to actively exchange ideas
and concepts. First, study the answers in your booklet and then EXPLAIN them to your group. Please don’t
just read them to your classmates, and don't let your classmates read their answers to you. In explaining
something to another person, most people gain a better understanding of it and often transmit a better
understanding. The pretest discussion and patient-oriented problem-solving parts of this activity are “open
book”. Be sure to refer to textbooks, notes, and other written resources whenever questions arise.

You will probably want to make notes on your pretest to help you review questions that you missed. Avoid
“collecting pages” for “later study and understanding”. Learn the concepts now so that later you will only
need to review them.

Reviewing pretest answers will probably take more time than any other part of this exercise. Some material
needed to solve this problem is not found in many textbooks and therefore has been incorporated into the
pretest answers.

(Definitions: Agglutination is “sticking” particles together. Hemagglutination is sticking RBCs together.
Antibody can “stick” things together and cause either precipitation if the antigen was previously in solution
or agglutination if the antigen was a suspension. Some, but not all, viruses can also cause
hemagglutination.)

1.     C is correct. This question is intended to demonstrate the theory behind agglutination. If a virus had
       no receptors for an RBC, it obviously could not stick to the RBC (Figure 1). If the virus had only
       one receptor, it could only bind to one RBC (Figure 2). If the virus had two or more receptors, it
       could bind to two or more RBCs and crosslink or agglutinate the RBCs (Figure 3).

       Figure 1                               Figure 2                               Figure 3




Virus with no receptor                Virus with one receptor                Virus with two (or more)
                                                                             receptors crosslinking

       Of course, ten receptors would work even better, but the question asks for the minimal number of
       binding sites a virus needs to agglutinate RBCs. In reality, influenza virus has about 550 receptors
       (hemagglutination molecules or binding sites) per virus particle.




                                                                                                      12
Influenza
Pretest Correct Answers
8.     B is correct. The definition of titer is still the reciprocal of the highest dilution giving the effect.
       This time, however, we are looking at HI.

12.    C is correct. Everybody should be able to read a graph like this, but the points we are trying to make
       are:
       1)      Before infection, little or no antibody is present.
       2)      It takes one to two weeks from the time of infection until enough antibody is produced to be
               detectable in the serum. Since the incubation period of influenza is one to three days, the
               antibody rise cannot be detected until about one week after the onset of symptoms.

       What is not evident from the graph is individual variation, especially in the amount of antibody
       produced. The time scale is fairly constant from one patient to another, but peak titers can vary from
       perhaps as low as 100 to thousands, or even higher.

       Incidentally, it is important to differentiate between “infection” and “illness”. Not all infections
       lead to illness. Some infections are “subclinical”, ie, the patient has no signs or symptoms of the
       disease even though the pathogen is replicating in his body and there is a subsequent rise in antibody
       to the pathogen.

14.    Can’t tell; therefore, C is correct. The HI test says nothing about the patient's present condition. It
       only measures his antibody level to a specific agglutinin or virus. He may have had the infection
       several years ago.




When your group has completed discussion of the pretest, you should have an understanding of the
principles involved in hemagglutination and hemagglutination inhibition. If you are still unclear about some
of these principles, be sure to consult textbooks or ask members of your group.




                                                                                                        13
Influenza
Instructions for the Clinical Problem
In the remainder of this package, you are to use your understanding of hemagglutination inhibition to answer
questions pertaining to an infectious disease epidemic. You will also discuss issues related to the diagnosis
and prevention of viral influenza.

Each of you has a complete copy of the same problem but only part of the data. After you have read the
problem, analyze your own data pool your knowledge with that of your groupmates and answer the
questions.


The Problem

There is an epidemic of upper respiratory disease at the Loving Care Orphanage. Of the 123 children in the
orphanage, three became ill during the last week of February. During the first week of March, 53 more
children became ill. “Flu shots” have never been used at this institution.

On March 5, a blood sample was taken from each of 12 children who were randomly selected from the 123
children at the Loving Care Orphanage. On March 26, another blood sample was drawn from the same 12
children.

Serum from these blood samples was assayed for antibody to two different viruses using the
hemagglutination inhibition (HI) assay. Your goal is to decide which influenza virus (A or B) is causing the
epidemic.




                                                                                                      14
Influenza
Review of Hemagglutination Inhibition (HI) Test

If a hemagglutinin (eg, influenza virus) is mixed with its homologous antibody in suitable amounts and
erythrocytes are then added, hemagglutination does not occur. Using this property, one can test the potency
of an antiserum to a virus hemagglutinin by measuring the minimum amount of antiserum (ie, highest
dilution of antiserum) necessary to completely inhibit hemagglutination.

The procedure is as follows:


Serial dilutions of serum

Prepare serial twofold dilutions of the serum to be tested for antibody to virus ranging from 1:10 to 1:640 in
0.5 ml amounts as shown below.




Begin with 1.0 ml of a 1:10 dilution of serum in well 1 and 0.5 ml of saline in wells 2 through 7. Remove
0.5 ml from well 1, place it in well 2, and mix. You now have 1.0 ml of a 1:20 dilution in well 2. Next, take
0.5 ml of the 1:20 dilution and mix it with the 0.5 ml of saline in well 3. This gives 1.0 ml of a 1:40 dilution
of serum in well 3 and leaves 0.5 ml of a 1:20 dilution in well 2. Repeat this procedure for wells 4, 5, 6, and
7 to give serial twofold dilutions in the row of wells. Ask your colleagues how to do serial fivefold
dilutions. (Answer: Transfer 0.1 ml into 0.4 ml.)


Addition of virus

Add 0.2 ml of the hemagglutinin (virus) suspension to each well, mix thoroughly, and incubate for 30
minutes at 37°C.


Addition of RBCs

Add 0.2 ml of washed chicken erythrocytes to each well and incubate for one hour at 37°C.




                                                                                                         15
Influenza
Controls

Three controls - virus, cells, and serum - are also set up.

1.     The virus control is made by mixing virus with red blood cells (RBCs) to check the
       hemagglutination capacity of the virus, ensuring no false results due to faulty virus.

2.     The serum control is set up by mixing a 1:10 dilution of antiserum with RBCs to see if “serum
       agglutinins” (ie, antibody in the human serum to chicken RBCs) are present. These agglutinins
       would give false hemagglutinations if present in high enough titer. Routinely, antisera are absorbed
       with chicken RBCs before being serially diluted in order to remove these interfering agglutinins. If
       this step is improperly performed, hemagglutination due to this antibody can be confused with viral
       hemagglutination, but the “serum control” will detect the error. You will see examples of this
       technical error in some of the data.

3.     Finally, a cell control is set up to determine whether or not the cells hemagglutinate when they are
       alone in saline and therefore give invalid results.

After the incubation period, the presence or absence of hemagglutination in each well should be recorded.
The highest dilution of serum causing complete inhibition of hemagglutination is taken as the endpoint. The
reciprocal of the highest dilution exhibiting HI is the titer.

To determine if there is evidence of acute infection one needs to observe an increase of at least fourfold in
the HI titer between two samples collected 2 - 3 weeks apart (acute and convalescent samples). Discuss
among yourselves why the titer needs to be at least fourfold higher to prove that a significant increase in
antibody titer has taken place.




                                                                                                      16
Influenza
Review of Hemagglutination Inhibition (HI) Test

If a hemagglutinin (eg, influenza virus) is mixed with its homologous antibody in suitable amounts and
erythrocytes are then added, hemagglutination does not occur. Using this property, one can test the potency
of an antiserum to a virus hemagglutinin by measuring the minimum amount of antiserum (ie, highest
dilution of antiserum) necessary to completely inhibit hemagglutination.

The procedure is as follows:


Serial dilutions of serum

Prepare serial twofold dilutions of the serum to be tested for antibody to virus ranging from 1:10 to 1:640 in
0.5 ml amounts as shown below.




Begin with 1.0 ml of a 1:10 dilution of serum in well 1 and 0.5 ml of saline in wells 2 through 7. Remove
0.5 ml from well 1, place it in well 2, and mix. You now have 1.0 ml of a 1:20 dilution in well 2. Next, take
0.5 ml of the 1:20 dilution and mix it with the 0.5 ml of saline in well 3. This gives 1.0 ml of a 1:40 dilution
of serum in well 3 and leaves 0.5 ml of a 1:20 dilution in well 2. Repeat this procedure for wells 4, 5, 6, and
7 to give serial twofold dilutions in the row of wells. Ask your colleagues how to do serial fivefold
dilutions. (Answer: Transfer 0.1 ml into 0.4 ml.)


Addition of virus

Add 0.2 ml of the hemagglutinin (virus) suspension to each well, mix thoroughly, and incubate for 30
minutes at 37°C.


Addition of RBCs

Add 0.2 ml of washed chicken erythrocytes to each well and incubate for one hour at 37°C.




                                                                                                         17
Influenza
Controls

Three controls - virus, cells, and serum - are also set up.

1.     The virus control is made by mixing virus with red blood cells (RBCs) to check the
       hemagglutination capacity of the virus, ensuring no false results due to faulty virus.

2.     The serum control is set up by mixing a 1:10 dilution of antiserum with RBCs to see if “serum
       agglutinins” (ie, antibody in the human serum to chicken RBCs) are present. These agglutinins
       would give false hemagglutinations if present in high enough titer. Routinely, antisera are absorbed
       with chicken RBCs before being serially diluted in order to remove these interfering agglutinins. If
       this step is improperly performed, hemagglutination due to this antibody can be confused with viral
       hemagglutination, but the “serum control” will detect the error. You will see examples of this
       technical error in some of the data.

3.     Finally, a cell control is set up to determine whether or not the cells hemagglutinate when they are
       alone in saline and therefore give invalid results.

After the incubation period, the presence or absence of hemagglutination in each well should be recorded.
The highest dilution of serum causing complete inhibition of hemagglutination is taken as the endpoint. The
reciprocal of the highest dilution exhibiting HI is the titer.

To determine if there is evidence of acute infection one needs to observe an increase of at least fourfold in
the HI titer between two samples collected 2 - 3 weeks apart (acute and convalescent samples). Discuss
among yourselves why the titer needs to be at least fourfold higher to prove that a significant increase in
antibody titer has taken place.




                                                                                                      18
Influenza
Review of Hemagglutination Inhibition (HI) Test

If a hemagglutinin (eg, influenza virus) is mixed with its homologous antibody in suitable amounts and
erythrocytes are then added, hemagglutination does not occur. Using this property, one can test the potency
of an antiserum to a virus hemagglutinin by measuring the minimum amount of antiserum (ie, highest
dilution of antiserum) necessary to completely inhibit hemagglutination.

The procedure is as follows:


Serial dilutions of serum

Prepare serial twofold dilutions of the serum to be tested for antibody to virus ranging from 1:10 to 1:640 in
0.5 ml amounts as shown below.




Begin with 1.0 ml of a 1:10 dilution of serum in well 1 and 0.5 ml of saline in wells 2 through 7. Remove
0.5 ml from well 1, place it in well 2, and mix. You now have 1.0 ml of a 1:20 dilution in well 2. Next, take
0.5 ml of the 1:20 dilution and mix it with the 0.5 ml of saline in well 3. This gives 1.0 ml of a 1:40 dilution
of serum in well 3 and leaves 0.5 ml of a 1:20 dilution in well 2. Repeat this procedure for wells 4, 5, 6, and
7 to give serial twofold dilutions in the row of wells. Ask your colleagues how to do serial fivefold
dilutions. (Answer: Transfer 0.1 ml into 0.4 ml.)


Addition of virus

Add 0.2 ml of the hemagglutinin (virus) suspension to each well, mix thoroughly, and incubate for 30
minutes at 37°C.


Addition of RBCs

Add 0.2 ml of washed chicken erythrocytes to each well and incubate for one hour at 37°C.




                                                                                                         19
Influenza
Controls

Three controls - virus, cells, and serum - are also set up.

1.     The virus control is made by mixing virus with red blood cells (RBCs) to check the
       hemagglutination capacity of the virus, ensuring no false results due to faulty virus.

2.     The serum control is set up by mixing a 1:10 dilution of antiserum with RBCs to see if “serum
       agglutinins” (ie, antibody in the human serum to chicken RBCs) are present. These agglutinins
       would give false hemagglutinations if present in high enough titer. Routinely, antisera are absorbed
       with chicken RBCs before being serially diluted in order to remove these interfering agglutinins. If
       this step is improperly performed, hemagglutination due to this antibody can be confused with viral
       hemagglutination, but the “serum control” will detect the error. You will see examples of this
       technical error in some of the data.

3.     Finally, a cell control is set up to determine whether or not the cells hemagglutinate when they are
       alone in saline and therefore give invalid results.

After the incubation period, the presence or absence of hemagglutination in each well should be recorded.
The highest dilution of serum causing complete inhibition of hemagglutination is taken as the endpoint. The
reciprocal of the highest dilution exhibiting HI is the titer.

To determine if there is evidence of acute infection one needs to observe an increase of at least fourfold in
the HI titer between two samples collected 2 - 3 weeks apart (acute and convalescent samples). Discuss
among yourselves why the titer needs to be at least fourfold higher to prove that a significant increase in
antibody titer has taken place.




                                                                                                      20
Influenza
Review of Hemagglutination Inhibition (HI) Test

If a hemagglutinin (eg, influenza virus) is mixed with its homologous antibody in suitable amounts and
erythrocytes are then added, hemagglutination does not occur. Using this property, one can test the potency
of an antiserum to a virus hemagglutinin by measuring the minimum amount of antiserum (ie, highest
dilution of antiserum) necessary to completely inhibit hemagglutination.

The procedure is as follows:


Serial dilutions of serum

Prepare serial twofold dilutions of the serum to be tested for antibody to virus ranging from 1:10 to 1:640 in
0.5 ml amounts as shown below.




Begin with 1.0 ml of a 1:10 dilution of serum in well 1 and 0.5 ml of saline in wells 2 through 7. Remove
0.5 ml from well 1, place it in well 2, and mix. You now have 1.0 ml of a 1:20 dilution in well 2. Next, take
0.5 ml of the 1:20 dilution and mix it with the 0.5 ml of saline in well 3. This gives 1.0 ml of a 1:40 dilution
of serum in well 3 and leaves 0.5 ml of a 1:20 dilution in well 2. Repeat this procedure for wells 4, 5, 6, and
7 to give serial twofold dilutions in the row of wells. Ask your colleagues how to do serial fivefold
dilutions. (Answer: Transfer 0.1 ml into 0.4 ml.)


Addition of virus

Add 0.2 ml of the hemagglutinin (virus) suspension to each well, mix thoroughly, and incubate for 30
minutes at 37°C.


Addition of RBCs

Add 0.2 ml of washed chicken erythrocytes to each well and incubate for one hour at 37°C.




                                                                                                         21
Influenza
Controls

Three controls - virus, cells, and serum - are also set up.

1.     The virus control is made by mixing virus with red blood cells (RBCs) to check the
       hemagglutination capacity of the virus, ensuring no false results due to faulty virus.

2.     The serum control is set up by mixing a 1:10 dilution of antiserum with RBCs to see if “serum
       agglutinins” (ie, antibody in the human serum to chicken RBCs) are present. These agglutinins
       would give false hemagglutinations if present in high enough titer. Routinely, antisera are absorbed
       with chicken RBCs before being serially diluted in order to remove these interfering agglutinins. If
       this step is improperly performed, hemagglutination due to this antibody can be confused with viral
       hemagglutination, but the “serum control” will detect the error. You will see examples of this
       technical error in some of the data.

3.     Finally, a cell control is set up to determine whether or not the cells hemagglutinate when they are
       alone in saline and therefore give invalid results.

After the incubation period, the presence or absence of hemagglutination in each well should be recorded.
The highest dilution of serum causing complete inhibition of hemagglutination is taken as the endpoint. The
reciprocal of the highest dilution exhibiting HI is the titer.

To determine if there is evidence of acute infection one needs to observe an increase of at least fourfold in
the HI titer between two samples collected 2 - 3 weeks apart (acute and convalescent samples). Discuss
among yourselves why the titer needs to be at least fourfold higher to prove that a significant increase in
antibody titer has taken place.




                                                                                                      22
Influenza

Data Sheet 1– Hemagglutination Inhibition Assay
Influenza B Virus
                                                                     CONTROLS
                                                                  Serum Saline Virus
        AGE                      DILUTION OF SERUM                   +    +      +
PATIENT (yrs) DATE 1:10   1:20    1:40 1:80 1:160 1:320   1:640    Cells Cells Cells




Influenza A Virus
                                                                     CONTROLS
                                                                  Serum Saline Virus
        AGE                  DILUTION OF SERUM                       +    +      +
PATIENT (yrs) DATE 1:10 1:20 1:40 1:80 1:160 1:320        1:640    Cells Cells Cells




                                                                                       23
Data Sheet 2– Hemagglutination Inhibition Assay

Influenza B Virus
                                                                                   CONTROLS
                                                                                Serum Saline Virus
        AGE                             DILUTION OF SERUM                          +     +      +
PATIENT (yrs) DATE 1:10          1:20    1:40 1:80 1:160 1:320         1:640     Cells Cells Cells




Influenza A Virus
                                                                                   CONTROLS
                                                                                Serum Saline Virus
        AGE                  DILUTION OF SERUM                                     +    +      +
PATIENT (yrs) DATE 1:10 1:20 1:40 1:80 1:160 1:320                     1:640     Cells Cells Cells




*IMPORTANT: In preparing the HI test on four year old patient E, influenza B and A on 3/26, the technician ran
            out of chicken erythrocytes and had to switch to a new batch of cells. This was the only test
            using these cells.


                                                                                                       24
Hemagglutination Inhibition Assay - Interpretation
Influenza B Virus

1. What are the titers of HI antibody in these six children?

Patient/Date                      Influenza B titer                 Influenza A titer
A    3/5
     3/26
B    3/5
     3/26
C    3/5
     3/26
D    3/5
     3/26
E    3/5
     3/26
H 3/5
     3/26

2. Were any of these patients infected at the time the first blood sample was collected? By which one
   of the influenza viruses?




3. Should any of the tests be considered invalid?




                                                                                              25
Influenza
                                      Influenza:
                                      Serologic Diagnosis
                                      and Epidemiology
                                      Marvin A. Cuchens, PhD
                                          Department of Microbiology
                                          University of Mississippi Medical Center
                                          Jackson, MS
                                      Bradley S. Bender, MD
                                          Department of Medicine
                                          University of Florida College of Medicine
                                      Steven Taffet, PhD
                                          Department of Microbiology and Immunology
                                          SUNY Upstate Medical University
                                          Syracuse, NY

                                      Originally Developed by:
                                      Parker A. Small, Jr., MD
                                      R. G. Weber, PhD
                                          Department of Immunology and Medical Microbiology
                                          College of Medicine, University of Florida
                                      Susan M. Johnson, PhD
                                          College of Pharmacy. University of Florida
                                          Gainesville, Florida

                                      Modified by:
                                      Jean-Michel Goust, M.D.
                                      Gabriel Virella, M.D., Ph.D.
                                          Medical University of South Carolina, Charleston, SC


                                            BOOK B
            Note to Students
                  The fundamental purpose of all activities in health-care professions is to help other people. Like
            all behavior, helping behavior becomes more effective and natural with practice. This workbook
            enables you to practice by helping your fellow students to learn basic science. Your skill at helping
            your fellow students should relate to your ability to help your patients in the future.
                  This is a Patient-Oriented Problem-Learning (“POPS”) workbook designed for four students.
            Before beginning this session, you should have (a) studied the objectives designed to prepare you for
            it, (b) taken the pretest, and (c) reviewed the topics listed at the end of the pretest. Now, each of you
            should take one of the four color-coded booklets and follow the directions in it. If your group has
            only three students, one of you should take two booklets. Leave the remainder of the workbook
            intact until you are given further instructions.
                  For additional information, SEE:
            http://www.medinfo.ufl.edu/cme/flu/flu.html
            http://www.cdc.gov/ncidod/diseases/flu/fluvirus.htm

                                      A Patient-Oriented Problem-Solving (POPS) System
                                      System supported by the American Medical Association Education and Research Foundation

                                                                                                                          26
Influenza
Introduction to the Patient-Oriented
Problem-Solving (POPS) System
This is a Patient-Oriented Problem-Solving activity. The purposes are:

1.    To help you learn how to apply your basic science knowledge to the solution of clinical problems

2.   To help you learn how to better use sources (ie, textbooks and peers) that will be available to you
     throughout your career

3.   To help you work with your fellow students and thus
     a. increase your ability to evaluate your colleagues’ opinions, thought processes, and diagnoses
     b. increase communications skills
     c. get to know your classmates better

This activity consists of four phases. First, you will review the attached set of objectives, do background
reading on the topics to be covered, and complete the pretest on your own. In the second phase, you will
join three other students and review the pretest answers in an “open-book” discussion. In the third phase,
the group will solve patient-oriented problems. Information exchange and group interaction are keys to the
success of this phase. This process will allow you to teach your fellow students and, at the same time, learn
from them. Finally, you will take a posttest, individually, which will enable you to assess your progress.




InfluenzaFinal02.gv                                                                                   27
Influenza
Introduction
Influenza is an epidemic disease that occurs yearly and causes much suffering and loss of life. Moreover,
at less frequent intervals (eg 1918, 1957, 1968 and 1977), influenza causes world pandemics; the one in
1918 caused 20 million deaths. This Patient-Oriented Problem-Solving activity deals with:
  • The laboratory procedures used to diagnose the disease (the basic concepts are also applicable to the
     diagnosis of many other diseases).
  • The measures that can be taken to prevent the disease in different populations.
  • The mechanism of action of the pharmacological agents that have activity against the influenza virus

Influenza virus and certain other viruses are able to agglutinate red blood cells (RBCs). This is called
hemagglutination (HA). However, in the presence of specific antibody to the virus, HA is blocked. This
phenomenon provides the basis for an assay used to detect and quantitate specific antiviral antibodies in
serum. This test is known as the hemagglutination inhibition (HI) assay.

In this activity, you will be asked to analyze HI data from several patients and to discuss other diagnostic
alternatives and preventive measures against influenza. Upon completion of the group session, you should
be able to:
1) determine, based upon HI data, whether a patient was
   a) infected with the pathogen during the period of acute illness or
   b) infected or immunized prior to that time with an antigenically identical or similar pathogen.
In order to accomplish these objectives, you must be able to:
2) schematically draw the in vitro interactions of virus, RBCs, and antibody in HA and HI assays.
3) interpret an HI assay, determine the HI titer, and defend or dispute the validity of the data based on
   the controls.
4) compare acute and convalescent serum titers to determine whether there has been a significant
   increase in antibody.
5) discuss other diagnostic procedures that can be used and when are they indicated
6) list the preventive measures that are available for influenza, discuss their advantages and limitations.
7) summarize the mechanisms of action of the different ant-viral agents that have activity against the
   influenza virus
In addition to learning these facts and concepts, it is important that you get to know your classmates better
and become more skilled at teaching and learning from each other. Much of your learning during your
clinical training and throughout your professional career will result from interactions with peers, house staff,
and practicing physicians. Improving your ability to be patient with, considerate of, and helpful to your
colleagues is easier now (when the “pressure” comes from a simulated clinical problem) than it will be when
the well-being of an actual patient is at stake. Good habits that you develop now hopefully will continue in
the future.


When you have become familiar with the objectives, complete the pretest on the next page.

InfluenzaFinal02.gv                                                                                      28
Influenza
Pretest Correct Answers
You have the answers to some of the 18 pretest questions, and other members of your group have the
remainder. This arrangement is designed to encourage all members of your group to actively exchange ideas
and concepts. First, study the answers in your booklet and then EXPLAIN them to your group. Please don't
just read them to your classmates, and don't let your classmates read their answers to you. In explaining
something to another person, most people gain a better understanding of it and often transmit a better
understanding. The pretest discussion and patient-oriented problem-solving parts of this activity are “open
book”. Be sure to refer to textbooks, notes, and other written resources whenever questions arise.
You will probably want to make notes on your pretest to help you review questions that you missed. Avoid
“collecting pages” for “later study and understanding”. Learn the concepts now so that later you will only
need to review them.
Reviewing pretest answers will probably take more time than any other part of this exercise. Some material
needed to solve this problem is not found in many textbooks and therefore has been incorporated into the
pretest answers.

2.      Answer A is correct. Even though you may not know what the symbols mean, the control should be
        normal (ie, not hemagglutinated). That should tell you that  means no hemagglutination. In this
        case, RBCs are not crosslinked and roll down the sides of the test tube to form a “pellet” in the
        bottom.




                                                                           (viewed from above)

        Hemagglutinated cells do not settle out into a pellet but instead form a layer covering the entire
        bottom of the test tube. This is because they stick to each other and cannot "roll" down to form a
        pellet.




                                                                           (viewed from above)

4.      E is correct. In tubes 1 through 4, the virus is coated with antibody so it can no longer cause
        hemagglutination (ie, the antibody has inhibited hemagglutination).

10.     The correct answer is D. A applies to ribavirin, B to interferons, C to adenine adenosine arabinoside
        (Ara A) and idoxuridine, and while a drug with the mechanism of action summarized in E would
        certainly affect the replication of influenza viruses, such drug has not yet been developed.

When your group has completed its discussion of the pretest, you should have an understanding of the principles
involved in hemagglutination and hemagglutination inhibition. If you are still unclear about some of these principles,
be sure to consult textbooks or ask members of your group.

                                                                                                              29
Influenza
Instructions for the Clinical Problem

In the remainder of this package, you are to use your knowledge of hemagglutination inhibition to answer
questions pertaining to an infectious disease epidemic.

Each of you has a complete copy of the same problem but only part of the data. After you have read the
problem, analyze your own data and try to decide the antibody titer each patient has to each virus and, also,
which patients were infected with which viruses. Then pool your knowledge with that of your groupmates
and answer the questions.


The Problem

There is an epidemic of upper respiratory disease at the Loving Care Orphanage. Of the 123 children in the
orphanage, three became ill during the last week of February. During the first week of March, 53 more
children became ill. “Flu shots” have never been used at this institution.

On March 5, a blood sample was taken from each of 12 children who were randomly selected from the 123
children at the Loving Care Orphanage. On March 26, another blood sample was drawn from the same 12
children.

Serum from these blood samples was assayed for antibody to two different viruses using the
hemagglutination inhibition (HI) assay. Your goal is to decide which influenza virus (A or B) is causing the
epidemic.




InfluenzaFinal02.gv                                                                                   30
Influenza
Data Interpretation
Influenza B Virus

1. What are the titers of HI antibody in these six children?

Patient/Date                         Influenza B titer                     Influenza A titer
A    3/5                             <10                                   <10
     3/26                            160                                   <10
B    3/5                             <10                                   <10
     3/26                            <10                                   <10
C    3/5                             <10                                   <10
     3/26                            <10                                   <10
D    3/5                             10                                    <10
     3/26                            80                                    <10
E    3/5                             <10                                   40
     3/26                            <10                                   <10
H 3/5                                20                                    <10
     3/26                            160                                   <10

2. Were any of these patients infected at the time the first blood sample was collected? By which one
   of the influenza viruses?
   • Patients A and D showed a significant rise in antibody titer to influenza B between 3/5 and 3/26;
       therefore it can be assumed that these patients were infected on 3/5 or shortly thereafter.
   • Patient H had two antibodies in his serum: a cross-reactive antibody that agglutinated red cells and
       an antibody that inhibited viral hemagglutination. The second antibody existed in greater titers than
       that of the first, so that titers can still be read, and this patient also had an acute infection by the
       influenza B virus.

3. Should any of the tests be considered invalid?
   • Serum B 3/5, serum E 3/26 and serum H (both dates) had antibodies that agglutinated the red cells
      (see controls); therefore those tests potentially not valid. However, for the reasons explained above,
      the results obtained with the two samples of serum H were valid as far as indicating an acute
      infection with influenza B virus.




InfluenzaFinal02.gv                                                                                     31
Influenza

Data Sheet 3– Summary of Hemagglutination Inhibition Assay

Patient/Date                       Influenza B titer                  Influenza A titer
A    3/5                           <10                                <10
     3/26                          160                                <10
B    3/5                           not valid                          not valid
     3/26                          <10                                <10
C    3/5                           <10                                <10
     3/26                          <10                                <10
D    3/5                           10                                 <10
     3/26                          80                                 <10
E    3/5                           <10                                40
     3/26                          not valid                          not valid
H 3/5                              20                                 not valid
     3/26                          160                                not valid
F    3/5                           <10                                40
     3/26                          >640                               40
G 3/5                              not valid                          80
     3/26                          not valid                          160
L    3/5                           not valid                          not valid
     3/26                          <10                                <10
I    3/5                           160                                20
     3/26                          80                                 40
J    3/5                           10                                 10
     3/26                          80                                 20
K 3/5                              <10                                not valid
     3/26                          <10                                20


1. Which patient(s) showed evidence of infection with influenza B virus shortly before or after 3/5?



2. Which patient(s) showed evidence of infection with influenza A virus before 3/5?



3. Which patient(s) never had an influenza A virus infection?



4. The epidemic was caused by which influenza virus? A? B? Both? Neither?




                                                                                                 32
Influenza

Group answers

1.   Patients A, D, H, F and J had significant elevations (≥ four fold increase) in the titers of HI
     antibodies to influenza B virus between 3/5 and 3/26.


2.   Patients with influenza A infections before 3/5:
     E, F, G, I, J (K is likely, but absence or invalidity of 3/5 data does not permit us to be certain.)


3.   Patients with no evidence of prior influenza A infections:
     A, B, C, D, L
     Patient B, 3/5, has not been infected with influenza A. Although the assay on 3/5 is invalid and
     hemagglutination is probably due to serum agglutinins plus virus, the data obtained on 3/26 is valid
     and indicates no anti-influenza A antibody. Since patient H had invalid assays on 3/5 and 3/26, it is
     not possible to determine whether or not he has ever had influenza A.

4.   Cause of current epidemic: Influenza B virus.
     Looking at the booklet D data sheet, it might appear that both influenza A and B viruses are the
     causative agents. However, the test is accurate within a range of up to + 100% or + 1 well;
     therefore, the one-well “increase” in titer on 3/26 on patients I and J is not significant. Put another
     way, if a patient has enough antibody to give a titer of 1:39, his 1:20 dilution would be positive but
     his 1:40 dilution would be negative. Therefore, the results would be indistinguishable from those of
     a patient with just enough antibody to give a titer of 1:20. Please be sure each group member
     understands this reasoning! A one-tube rise does not mean a doubling of the antibody level and
     cannot be considered proof of an infection.




                                                                                                     33
Influenza
                                      Influenza:
                                      Serologic Diagnosis
                                      and Epidemiology
                                      Marvin A. Cuchens, PhD
                                          Department of Microbiology
                                          University of Mississippi Medical Center
                                          Jackson, MS
                                      Bradley S. Bender, MD
                                          Department of Medicine
                                          University of Florida College of Medicine
                                      Steven Taffet, PhD
                                          Department of Microbiology and Immunology
                                          SUNY Upstate Medical University
                                          Syracuse, NY

                                      Originally Developed by:
                                      Parker A. Small, Jr., MD
                                      R. G. Weber, PhD
                                          Department of Immunology and Medical Microbiology
                                          College of Medicine, University of Florida
                                      Susan M. Johnson, PhD
                                          College of Pharmacy. University of Florida
                                          Gainesville, Florida

                                      Modified by:
                                      Jean-Michel Goust, M.D.
                                      Gabriel Virella, M.D., Ph.D.
                                          Medical University of South Carolina, Charleston, SC


                                           BOOK C
            Note to Students
                  The fundamental purpose of all activities in health-care professions is to help other people. Like
            all behavior, helping behavior becomes more effective and natural with practice. This workbook
            enables you to practice by helping your fellow students to learn basic science. Your skill at helping
            your fellow students should relate to your ability to help your patients in the future.
                  This is a Patient-Oriented Problem-Learning (“POPS”) workbook designed for four students.
            Before beginning this session, you should have (a) studied the objectives designed to prepare you for
            it, (b) taken the pretest, and (c) reviewed the topics listed at the end of the pretest. Now, each of you
            should take one of the four color-coded booklets and follow the directions in it. If your group has
            only three students, one of you should take two booklets. Leave the remainder of the workbook
            intact until you are given further instructions.
                  For additional information, SEE:
            http://www.medinfo.ufl.edu/cme/flu/flu.html
            http://www.cdc.gov/ncidod/diseases/flu/fluvirus.htm
                                      A Patient-Oriented Problem-Solving (POPS) System
                                      System supported by the American Medical Association Education and Research Foundation


                                                                                                                          34
Influenza
This is a Patient-Oriented Problem-Solving activity. The purposes are:

1.   To help you learn how to apply your basic science knowledge to the solution of clinical problems

2.   To help you learn how to better use sources (ie, textbooks and peers) that will be available to you
     throughout your career

3.   To help you work with your fellow students and thus
     a. increase your ability to evaluate your colleagues’ opinions, thought processes, and diagnoses
     b. increase communications skills
     c. get to know your classmates better

This activity consists of four phases. First, you will review the attached set of objectives, do background
reading on the topics to be covered, and complete the pretest on your own. In the second phase, you will
join three other students and review the pretest answers in an “open-book” discussion. In the third phase,
the group will solve patient-oriented problems. Information exchange and group interaction are keys to the
success of this phase. This process will allow you to teach your fellow students and, at the same time, learn
from them. Finally, you will take a posttest, individually, which will enable you to assess your progress.




                                                                                                      35
Influenza
Introduction
Influenza is an epidemic disease that occurs yearly and causes much suffering and loss of life. Moreover,
at less frequent intervals (eg 1918, 1957, 1968 and 1977), influenza causes world pandemics; the one in
1918 caused 20 million deaths. This Patient-Oriented Problem-Solving activity deals with:
  • The laboratory procedures used to diagnose the disease (the basic concepts are also applicable to the
     diagnosis of many other diseases).
  • The measures that can be taken to prevent the disease in different populations.
  • The mechanism of action of the pharmacological agents that have activity against the influenza virus

Influenza virus and certain other viruses are able to agglutinate red blood cells (RBCs). This is called
hemagglutination (HA). However, in the presence of specific antibody to the virus, HA is blocked. This
phenomenon provides the basis for an assay used to detect and quantitate specific antiviral antibodies in
serum. This test is known as the hemagglutination inhibition (HI) assay.

In this activity, you will be asked to analyze HI data from several patients and to discuss other diagnostic
alternatives and preventive measures against influenza. Upon completion of the group session, you should
be able to:
1) determine, based upon HI data, whether a patient was
   a) infected with the pathogen during the period of acute illness or
   b) infected or immunized prior to that time with an antigenically identical or similar pathogen.
In order to accomplish these objectives, you must be able to:
2) schematically draw the in vitro interactions of virus, RBCs, and antibody in HA and HI assays.
3) interpret an HI assay, determine the HI titer, and defend or dispute the validity of the data based on
   the controls.
4) compare acute and convalescent serum titers to determine whether there has been a significant
   increase in antibody.
5) discuss other diagnostic procedures that can be used and when are they indicated
6) list the preventive measures that are available for influenza, discuss their advantages and limitations.
7) summarize the mechanisms of action of the different ant-viral agents that have activity against the
   influenza virus
In addition to learning these facts and concepts, it is important that you get to know your classmates better
and become more skilled at teaching and learning from each other. Much of your learning during your
clinical training and throughout your professional career will result from interactions with peers, house staff,
and practicing physicians. Improving your ability to be patient with, considerate of, and helpful to your
colleagues is easier now (when the “pressure” comes from a simulated clinical problem) than it will be when
the well-being of an actual patient is at stake. Good habits that you develop now hopefully will continue in
the future.




                                                                                                         36
Influenza
Pretest Correct Answers

       You have the answers to some of the 15 pretest questions, and other members of your group have the
       remainder. This arrangement is designed to encourage all members of your group to actively
       exchange ideas and concepts. First, study the answers in your booklet and then EXPLAIN them to
       your group. Please don't just read them to your classmates, and don't let your classmates read their
       answers to you. In explaining something to another person, most people gain a better understanding
       of it and often transmit a better understanding. The pretest discussion and patient-oriented problem-
       solving parts of this activity are “open book”. Be sure to refer to textbooks, notes, and other written
       resources whenever questions arise.

       You will probably want to make notes on your pretest to help you review questions that you missed.
       Avoid “collecting pages” for “later study and understanding”. Learn the concepts now so that later
       you will only need to review them.

4.     E is the incorrect answer. Influenza can be cultured from respiratory secretions, but results will
        not be available for several days. There is a rapid antigen detection system that can be
        performed on nasal or throat swabs; a result is available within one hour. An IgM titer to a
        specific pathogen can frequently occur and can be obtained, but a detection kit is not
        commercially available for anti-IgM antibodies against influenza viruses. Many infectious
        diseases can be diagnosed by a 4-fold change in IgG titers; the limitation of this method is that
        the convalescent serum is obtained about two weeks after the infection, so after processing,
        results are frequently not available until one month after the patient is seen!

9.     C is correct. Oseltamivir , given orally twice a day, has been approved for the prevention of
        influenza A and B. Acyclovir and gancyclovir are used for the treatment of infections with
        viruses of the Herpes group. Ribavirin is moderately effective against several RNA viruses, but
        not against orthomyxoviruses. Zydovudine is effective against HIV.

11.    D is correct. The influenza vaccine is an inactivated vaccine, prepared with the most prevalent
        strains of influenza A and B virus. The measles-mumps and rubella vaccines are examples of
        attenuated vaccines. The hepatitis B vaccine is an example of a (recombinant) component
        vaccine. Vaccines based on recombinant organisms are in the experimental stages.

When your group has completed its discussion of the pretest, you should have an understanding of the
principles involved in hemagglutination and hemagglutination inhibition. If you are still unclear about
some of these principles, be sure to consult textbooks or ask members of your group.




                                                                                                       37
Influenza
Instructions for the Clinical Problem

In the remainder of this package, you are to use your knowledge about prophylaxis of influenza to
answer (as a group) questions related to clinical situations in which preventive measures could have has
a significant impact.


Case scenario # 1

There is an epidemic of upper respiratory disease at the Loving Care Orphanage. Of the 123 children in
the orphanage, three became ill during the last week of February. During the first week of March, 53
more children became ill. “Flu shots” have never been used at this institution.

Child M, a 7-yr old male who in the past suffered recurrent otitis and pneumonia became so ill that he
had to be admitted to the Intensive Care unit of the local hospital, and eventually died.

The HI antibody titer performed in this child at the time of admission and 7 days later showed titers of
less than 10 for both influenza A and influenza B viruses. Additional investigations revealed that this
child had a mild form of X-linked agammaglobulinemia.

What measures could have been taken when the epidemic started to prevent the infection of this child?




                                                                                                    38
Influenza

Prevention

Influenza infections can be prevented by immunization or with antiviral drugs. Immunization is the most
cost-effective approach, but it may not have been of great benefit for patient M because of his humoral
immunodeficiency.

Several antiviral drugs have been shown to prevent viral influenza. Amantadine and rimantadine, drugs
that block the release of viral nucleocapsids in the cytoplasm of non-infected cells, are mostly effective
against influenza A, and so they may not have been of great use in child M because the epidemic in the
orphanage was caused by influenza B virus.

Two newer antivirals, zanamivir [Relenza] and Oseltamivir are effective in preventing infection by both
viruses. Zanamavir is administered by inhalation and oseltamivir is given orally. Both drugs are
neuraminidase inhibitors and prevent the spread of influenza viruses to non-infected cells. Both drugs
are effective against A and B influenza viruses.

In a case of an epidemic such as the one described in this POPS, the best approach to protect the non-
infected children is starting one of the new antivirals for immediate protection and start the
immunization schedule at the same time to induce long term protection, which involves both the
synthesis of protective antibodies and the stimulation of cell mediated immunity against the virus




                                                                                                     39
Influenza
                                      Influenza:
                                      Serologic Diagnosis
                                      and Epidemiology
                                      Marvin A. Cuchens, PhD
                                          Department of Microbiology
                                          University of Mississippi Medical Center
                                          Jackson, MS
                                      Bradley S. Bender, MD
                                          Department of Medicine
                                          University of Florida College of Medicine
                                      Steven Taffet, PhD
                                          Department of Microbiology and Immunology
                                          SUNY Upstate Medical University
                                          Syracuse, NY

                                      Originally Developed by:
                                      Parker A. Small, Jr., MD
                                      R. G. Weber, PhD
                                          Department of Immunology and Medical Microbiology
                                          College of Medicine, University of Florida
                                      Susan M. Johnson, PhD
                                          College of Pharmacy. University of Florida
                                          Gainesville, Florida

                                      Modified by:
                                      Jean-Michel Goust, M.D.
                                      Gabriel Virella, M.D., Ph.D.
                                          Medical University of South Carolina, Charleston, SC


                                           BOOK D
            Note to Students
                  The fundamental purpose of all activities in health-care professions is to help other people. Like
            all behavior, helping behavior becomes more effective and natural with practice. This workbook
            enables you to practice by helping your fellow students to learn basic science. Your skill at helping
            your fellow students should relate to your ability to help your patients in the future.
                  This is a Patient-Oriented Problem-Learning (“POPS”) workbook designed for four students.
            Before beginning this session, you should have (a) studied the objectives designed to prepare you for
            it, (b) taken the pretest, and (c) reviewed the topics listed at the end of the pretest. Now, each of you
            should take one of the four color-coded booklets and follow the directions in it. If your group has
            only three students, one of you should take two booklets. Leave the remainder of the workbook
            intact until you are given further instructions.
                  For additional information, SEE:
            http://www.medinfo.ufl.edu/cme/flu/flu.html
            http://www.cdc.gov/ncidod/diseases/flu/fluvirus.htm
                                      A Patient-Oriented Problem-Solving (POPS) System
                                      System supported by the American Medical Association Education and Research Foundation


                                                                                                                          40
Influenza
This is a Patient-Oriented Problem-Solving activity. The purposes are:

1.   To help you learn how to apply your basic science knowledge to the solution of clinical problems

2.   To help you learn how to better use sources (ie, textbooks and peers) that will be available to you
     throughout your career

3.   To help you work with your fellow students and thus
     a. increase your ability to evaluate your colleagues’ opinions, thought processes, and diagnoses
     b. increase communications skills
     c. get to know your classmates better

This activity consists of four phases. First, you will review the attached set of objectives, do background
reading on the topics to be covered, and complete the pretest on your own. In the second phase, you will
join three other students and review the pretest answers in an “open-book” discussion. In the third phase,
the group will solve patient-oriented problems. Information exchange and group interaction are keys to the
success of this phase. This process will allow you to teach your fellow students and, at the same time, learn
from them. Finally, you will take a posttest, individually, which will enable you to assess your progress.




                                                                                                      41
Influenza
Introduction
Influenza is an epidemic disease that occurs yearly and causes much suffering and loss of life. Moreover,
at less frequent intervals (eg 1918, 1957, 1968 and 1977), influenza causes world pandemics; the one in
1918 caused 20 million deaths. This Patient-Oriented Problem-Solving activity deals with:
  • The laboratory procedures used to diagnose the disease (the basic concepts are also applicable to the
     diagnosis of many other diseases).
  • The measures that can be taken to prevent the disease in different populations.
  • The mechanism of action of the pharmacological agents that have activity against the influenza virus

Influenza virus and certain other viruses are able to agglutinate red blood cells (RBCs). This is called
hemagglutination (HA). However, in the presence of specific antibody to the virus, HA is blocked. This
phenomenon provides the basis for an assay used to detect and quantitate specific antiviral antibodies in
serum. This test is known as the hemagglutination inhibition (HI) assay.

In this activity, you will be asked to analyze HI data from several patients and to discuss other diagnostic
alternatives and preventive measures against influenza. Upon completion of the group session, you should
be able to:
1) determine, based upon HI data, whether a patient was
   a) infected with the pathogen during the period of acute illness or
   b) infected or immunized prior to that time with an antigenically identical or similar pathogen.
In order to accomplish these objectives, you must be able to:
2) schematically draw the in vitro interactions of virus, RBCs, and antibody in HA and HI assays.
3) interpret an HI assay, determine the HI titer, and defend or dispute the validity of the data based on
   the controls.
4) compare acute and convalescent serum titers to determine whether there has been a significant
   increase in antibody.
5) discuss other diagnostic procedures that can be used and when are they indicated
6) list the preventive measures that are available for influenza, discuss their advantages and limitations.
7) summarize the mechanisms of action of the different ant-viral agents that have activity against the
   influenza virus
In addition to learning these facts and concepts, it is important that you get to know your classmates better
and become more skilled at teaching and learning from each other. Much of your learning during your
clinical training and throughout your professional career will result from interactions with peers, house staff,
and practicing physicians. Improving your ability to be patient with, considerate of, and helpful to your
colleagues is easier now (when the “pressure” comes from a simulated clinical problem) than it will be when
the well-being of an actual patient is at stake. Good habits that you develop now hopefully will continue in
the future.




                                                                                                         42
Influenza
Pretest Correct Answers
You have the answers to some of the 18 pretest questions, and other members of your group have the
remainder. This arrangement is designed to encourage all members of your group to actively exchange ideas
and concepts. First, study the answers in your booklet and then EXPLAIN them to your group. Please don't
just read them to your classmates, and don't let your classmates read their answers to you. In explaining
something to another person, most people gain a better understanding of it and often transmit a better
understanding. The pretest discussion and patient-oriented problem-solving parts of this activity are “open
book”. Be sure to refer to textbooks, notes, and other written resources whenever questions arise.
You will probably want to make notes on your pretest to help you review questions that you missed. Avoid
“collecting pages” for “later study and understanding”. Learn the concepts now so that later you will only
need to review them.
Reviewing pretest answers will probably take more time than any other part of this exercise. Some material
needed to solve this problem is not found in many textbooks and therefore has been incorporated into the
pretest answers.
3.     C is correct. Tube #1 is undiluted; tube #2 is diluted 1:2; tube #3, 1:4; tube #4, 1:8; and tube #5,
       1:16 (ie, 2-4). Ask your groupmates how they would do tenfold dilutions and what the titer would be
       in tube #5 if tenfold dilutions had been done. (Answer: Add 0.1 ml of sample to 0.9 ml of saline.
       The dilution would be 1:10,000, ie, 10-4.)
5.     All of the answers are correct except “D.” Influenza vaccine is recommended for persons at high
       risk for contracting influenza; to obtain a list of the high risk groups see this LINK
       (http://www.medinfo.ufl.edu/cme/flu/flu.html ). Serum anti-influenza antibody is induced against
       the surface antigens hemagglutinin and neuraminidase. These undergo minor (antigenic drift)
       changes each year. About every 10-20 years, influenza virus undergoes a major change in its
       antigenic structure (antigenic shift). Unlike antibody, which is principally involved in preventing
       infections, the main role of cellular immunity is to enhance recovery from viral infections. There is
       a clear decline in cell-mediated immunity in older persons and this helps explain why they have
       higher morbidity and mortality. While inactivated vaccines such as the current influenza vaccine
       induce serum IgG antibody, they are relatively poor inducers of cell-mediated immunity. Note that
       live virus vaccines can induce cell-mediated immunity.
7.     D is correct. Note that antibody is “coating” the virus so the virus cannot attach to the RBC.
13.    E is correct. This is another fairly obvious question that is intended to emphasize the persistence of
       antibody. Unfortunately, this does not occur with all infections or immunizations. Some lead to
       shorter persistence of antibody. Generally, live vaccines lead to more prolonged persistence of
       antibody than do nonliving vaccines (eg, tetanus toxoid).
15.    This is true; therefore, A is correct. This is a “brilliant qualitative summation” of the data presented.
       Ask the others in your group what the actual titer is. (Answer: They should agree with you that it is
       80.)
When your group has completed its discussion of the pretest, you should have an understanding of the
principles involved in hemagglutination and hemagglutination-inhibition. If you are still unclear about some
of these principles, be sure to consult textbooks or ask members of your group.


                                                                                                         43
Influenza
Instructions for the Clinical Problem

In the remainder of this package, you are to use your knowledge about prophylaxis of influenza to answer
(as a group) questions related to clinical situations in which preventive measures could have has a significant
impact.


Case scenario # 2

Mr. B., a sprightly 77 year-old man, came to visit his wife at the Golden Homes (a Retirement and
assisted living facility) after Thanksgiving 2000. He had dinner with many other guests and socialized
eagerly with them.

He started complaining of feeling achy and febrile that evening. His physician requested rapid testing for
Influenza A that came back positive. The Medical staff of the Golden Homes is very concerned: only a
third of the guests were vaccinated because of a delay in vaccine supply. In addition, 5 of the vaccinated
residents are already experiencing fever and respiratory symptoms.

It is decided that rapid testing would not be indicated because specificity and sensitivity vary depending
on the test manufacturer and because the test will not give any indication about whether the particular
strain in this outbreak has already undergone antigenic drift, since the epidemic started 4 weeks ago in
the state.

What procedure(s) should be ordered to determine the antigenic make-up of the virus involved in this
outbreak?
_________ PCR on throat washing
_________ CD4 counts
_________ Chest X rays
_________ Viral cultures and serological studies




                                                                                                        44
Influenza

Epidemiology

Viral cultures will allow to determine which Influenza virus ( A or B) is involved in the outbreak and
will also allow to determine wether the virus (particularly if it is Influenza A as identified in Mr B.) has
already drifted.

What is antigenic shift?

What is antigenic drift?

Why are these phenomena of concern to physicians?




                                                                                                      45
Influenza

Epidemiology & Prevention

Antigenic shift usually results from reassortment of genetic material between two different types of
influenza virus and causes the emergence of a totally new strain against which the population has little
protective immunity. Such events are usually followed by pandemics affecting large numbers of people
world-wide.

Antigenic drift results from point mutations in the viral hemagglutinins that result in loss of efficiency of
previously existing protective antibodies (the antibodies do not bind with the same affinity to mutant
epitopes). This is a cause of concern because vaccination loses its protective effects as the virus
continues to drift.

Viral cultures from the affected individuals and 20 still healthy residents came back 2 weeks later and
confirmed the existence of drift without shift and the data were sent to the CDC.

What would be the best course of action at this time?

_____ No further action needed

_____ Start mass immunization of all patients and staff

_____ Start administration of amantadine to patients and staff

_____ Start administration of oseltamivir to patients and staff




                                                                                                      46
Influenza

Prevention

Extract from "Prevention and Control of Influenza" MMWR April 20 2001
                       :
“When institutional outbreaks occur, chemoprophylaxis should be administered to all residents
regardless of whether they received influenza vaccination the previous fall and should be continued for
at least 2 weeks or 1 week after the end of the outbreak. It can also be offered to the unvaccinated staff “

http://www.cdc.gov/ncidod/diseases /flu/fuvirus.htm

Because oseltamivir is the only anti-viral approved by the FDA for the prevention of influenza A and B,
it should be given immediately to all patients and staff. Immunization could also be started, although its
protective effects may be severely limited by the fact that the virus responsible for the outbreak has
drifted relative to the strain included in the vaccine.




                                                                                                      47
Influenza
Posttest
Select the best answer for each question. Please mark your answers on this exam to facilitate discussion and
later review. If your instructor has provided a separate answer form, be sure you have identified yourself on
the form, then begin your answers with question 1. Mark your answers both on the form and on this exam.
Only one answer is correct.

For questions 1 through 3, both of the swine influenza hemagglutination inhibition (HI) assays shown
below were done on serum from one patient.




1.     The patient's HI serum titer on 1/12 was
(A)    <10
(B)    10
(C)    >640
(D)    640
(E)    None of these

2.     The patient's HI serum titer on 2/15 was
(A)    <10
(B)    10
(C)    80
(D)    160
(E)    None of these

3.     Which statement applies to the January-February period?
(A)    The patient had swine flu.
(B)    The patient had a swine flu immunization.
(C)    The patient either had swine flu or a flu immunization.
(D)    The patient had neither swine flu nor swine flu immunization.




                                                                                                      48
Influenza

4.       On November 6, a patient had the onset of an illness characterized by fever, chills, headache,
cough, and chest pain. The illness lasted one week. On December 5, she had another illness very similar to
the first, which lasted six days. She had no flu immunization during this period. Her HI titer to swine flu
virus was:
Nov. 6          10
Nov. 30         10
Dec. 20         160
(There were no laboratory errors.)

Select the valid conclusion from this data:
(A)     The patient was ill with swine flu on 11/6.
(B)     The patient was ill with swine flu later, and the 11/6 illness was due to another pathogen.
(C)     The patient was ill with swine flu on 12/20.
(D)     None of the above.

5.     A patient's serum titer was read as 80 initially and as 160 one month later in a test that used serial
twofold dilutions both times. Choose the most accurate interpretation of this data.
(A)    The amount of antibody in his serum has doubled.
(B)    The amount of antibody in his serum has increased between a few percent and almost fourfold.
(C)    The amount of antibody in his serum has increased between a few percent and 100%.
(D)    None of the above.

6.     A major problem encountered with the current influenza vaccine is:
(A)    Induction of neurological complications in about 1:10000 vaccinees
(B)    Loss of efficiency due to influenza virus drift
(C)    Poor immunogenicity of the viral polysaccharide used to prepare the vaccine
(D)    Possible infection of immunocompromised individuals
(E)    Reversion to disease-causing wild type

7.     The mechanism of action of zanamavir and oseltamavir involves:
(A)    Activation of the viral neuraminidase
(B)    Inhibition of the endonuclease that cuts the "cap" from nascent mammalian mRNA
(C)    Inhibition of the release of viral nucleocapsids into the cytoplasm of non-infected cells
(D)    Inhibition of viral propagation to non-infected cells
(E)    Interference with the viral protease




                                                                                                      49
Influenza
Questions 8 through 11 refer to the following HI pattern:




Using:

as symbol for red blood cell (RBC)


as symbol for antibody (Ab)


as symbol for virus

Match the following drawings with the wells. Each answer may only be used once.




8.       Is the 1:10 well represented by drawing       a, b, c, d, or e?

9.       Is the 1:80 well represented by drawing       a, b, c, d, or e?

10.      Is the 1:320 well represented by drawing      a, b, c, d, or e?

11.      Is the virus control represented by drawing a, b, c, d, or e?

When you have finished the posttest, discuss your answers with your colleagues and then compare them
with the correct answers. (See last pages in the workbook, or follow your instructor’s directions.)




                                                                                              50
Influenza
Posttest
Select the best answer for each question. Please mark your answers on this exam to facilitate discussion and
later review. If your instructor has provided a separate answer form, be sure you have identified yourself on
the form, then begin your answers with question 1. Mark your answers both on the form and on this exam.
Only one answer is correct.

For questions 1 through 3, both of the swine influenza hemagglutination inhibition (HI) assays shown
below were done on serum from one patient.




1.     The patient's HI serum titer on 1/12 was
(A)    <10
(B)    10
(C)    >640
(D)    640
(E)    None of these

2.     The patient's HI serum titer on 2/15 was
(A)    <10
(B)    10
(C)    80
(D)    160
(E)    None of these

3.     Which statement applies to the January-February period?
(A)    The patient had swine flu.
(B)    The patient had a swine flu immunization.
(C)    The patient either had swine flu or a flu immunization.
(D)    The patient had neither swine flu nor swine flu immunization.




                                                                                                      51
Influenza

4.       On November 6, a patient had the onset of an illness characterized by fever, chills, headache,
cough, and chest pain. The illness lasted one week. On December 5, she had another illness very similar to
the first, which lasted six days. She had no flu immunization during this period. Her HI titer to swine flu
virus was:
Nov. 6          10
Nov. 30         10
Dec. 20         160
(There were no laboratory errors.)

Select the valid conclusion from this data:
(A)     The patient was ill with swine flu on 11/6.
(B)     The patient was ill with swine flu later, and the 11/6 illness was due to another pathogen.
(C)     The patient was ill with swine flu on 12/20.
(D)     None of the above.

5.     A patient's serum titer was read as 80 initially and as 160 one month later in a test that used serial
twofold dilutions both times. Choose the most accurate interpretation of this data.
(A)    The amount of antibody in his serum has doubled.
(B)    The amount of antibody in his serum has increased between a few percent and almost fourfold.
(C)    The amount of antibody in his serum has increased between a few percent and 100%.
(D)    None of the above.

6.     A major problem encountered with the current influenza vaccine is:
(A)    Induction of neurological complications in about 1:10000 vaccinees
(B)    Loss of efficiency due to influenza virus drift
(C)    Poor immunogenicity of the viral polysaccharide used to prepare the vaccine
(D)    Possible infection of immunocompromised individuals
(E)    Reversion to disease-causing wild type

7.     The mechanism of action of zanamavir and oseltamavir involves:
(A)    Activation of the viral neuraminidase
(B)    Inhibition of the endonuclease that cuts the "cap" from nascent mammalian mRNA
(C)    Inhibition of the release of viral nucleocapsids into the cytoplasm of non-infected cells
(D)    Inhibition of viral propagation to non-infected cells
(E)    Interference with the viral protease




                                                                                                      52
Influenza
Questions 8 through 11 refer to the following HI pattern:




Using:

as symbol for red blood cell (RBC)


as symbol for antibody (Ab)


as symbol for virus

Match the following drawings with the wells. Each answer may only be used once.




8.       Is the 1:10 well represented by drawing       a, b, c, d, or e?

9.       Is the 1:80 well represented by drawing       a, b, c, d, or e?

10.      Is the 1:320 well represented by drawing      a, b, c, d, or e?

11.      Is the virus control represented by drawing a, b, c, d, or e?

When you have finished the posttest, discuss your answers with your colleagues and then compare them
with the correct answers. (See last pages in the workbook, or follow your instructor’s directions.)




                                                                                              53
Influenza
Posttest
Select the best answer for each question. Please mark your answers on this exam to facilitate discussion and
later review. If your instructor has provided a separate answer form, be sure you have identified yourself on
the form, then begin your answers with question 1. Mark your answers both on the form and on this exam.
Only one answer is correct.

For questions 1 through 3, both of the swine influenza hemagglutination inhibition (HI) assays shown
below were done on serum from one patient.




1.     The patient's HI serum titer on 1/12 was
(A)    <10
(B)    10
(C)    >640
(D)    640
(E)    None of these

2.     The patient's HI serum titer on 2/15 was
(A)    <10
(B)    10
(C)    80
(D)    160
(E)    None of these

3.     Which statement applies to the January-February period?
(A)    The patient had swine flu.
(B)    The patient had a swine flu immunization.
(C)    The patient either had swine flu or a flu immunization.
(D)    The patient had neither swine flu nor swine flu immunization.




                                                                                                      54
Influenza

4.       On November 6, a patient had the onset of an illness characterized by fever, chills, headache,
cough, and chest pain. The illness lasted one week. On December 5, she had another illness very similar to
the first, which lasted six days. She had no flu immunization during this period. Her HI titer to swine flu
virus was:
Nov. 6          10
Nov. 30         10
Dec. 20         160
(There were no laboratory errors.)

Select the valid conclusion from this data:
(A)     The patient was ill with swine flu on 11/6.
(B)     The patient was ill with swine flu later, and the 11/6 illness was due to another pathogen.
(C)     The patient was ill with swine flu on 12/20.
(D)     None of the above.

5.     A patient's serum titer was read as 80 initially and as 160 one month later in a test that used serial
twofold dilutions both times. Choose the most accurate interpretation of this data.
(A)    The amount of antibody in his serum has doubled.
(B)    The amount of antibody in his serum has increased between a few percent and almost fourfold.
(C)    The amount of antibody in his serum has increased between a few percent and 100%.
(D)    None of the above.

6.     A major problem encountered with the current influenza vaccine is:
(A)    Induction of neurological complications in about 1:10000 vaccinees
(B)    Loss of efficiency due to influenza virus drift
(C)    Poor immunogenicity of the viral polysaccharide used to prepare the vaccine
(D)    Possible infection of immunocompromised individuals
(E)    Reversion to disease-causing wild type

7.     The mechanism of action of zanamavir and oseltamavir involves:
(A)    Activation of the viral neuraminidase
(B)    Inhibition of the endonuclease that cuts the "cap" from nascent mammalian mRNA
(C)    Inhibition of the release of viral nucleocapsids into the cytoplasm of non-infected cells
(D)    Inhibition of viral propagation to non-infected cells
(E)    Interference with the viral protease




                                                                                                      55
Influenza
Questions 8 through 11 refer to the following HI pattern:




Using:

as symbol for red blood cell (RBC)


as symbol for antibody (Ab)


as symbol for virus

Match the following drawings with the wells. Each answer may only be used once.




8.       Is the 1:10 well represented by drawing       a, b, c, d, or e?

9.       Is the 1:80 well represented by drawing       a, b, c, d, or e?

10.      Is the 1:320 well represented by drawing      a, b, c, d, or e?

11.      Is the virus control represented by drawing a, b, c, d, or e?

When you have finished the posttest, discuss your answers with your colleagues and then compare them
with the correct answers. (See last pages in the workbook, or follow your instructor’s directions.)




                                                                                              56
Influenza
Posttest
Select the best answer for each question. Please mark your answers on this exam to facilitate discussion and
later review. If your instructor has provided a separate answer form, be sure you have identified yourself on
the form, then begin your answers with question 1. Mark your answers both on the form and on this exam.
Only one answer is correct.

For questions 1 through 3, both of the swine influenza hemagglutination inhibition (HI) assays shown
below were done on serum from one patient.




1.     The patient's HI serum titer on 1/12 was
(A)    <10
(B)    10
(C)    >640
(D)    640
(E)    None of these

2.     The patient's HI serum titer on 2/15 was
(A)    <10
(B)    10
(C)    80
(D)    160
(E)    None of these

3.     Which statement applies to the January-February period?
(A)    The patient had swine flu.
(B)    The patient had a swine flu immunization.
(C)    The patient either had swine flu or a flu immunization.
(D)    The patient had neither swine flu nor swine flu immunization.




                                                                                                      57
Influenza

4.       On November 6, a patient had the onset of an illness characterized by fever, chills, headache,
cough, and chest pain. The illness lasted one week. On December 5, she had another illness very similar to
the first, which lasted six days. She had no flu immunization during this period. Her HI titer to swine flu
virus was:
Nov. 6          10
Nov. 30         10
Dec. 20         160
(There were no laboratory errors.)

Select the valid conclusion from this data:
(A)     The patient was ill with swine flu on 11/6.
(B)     The patient was ill with swine flu later, and the 11/6 illness was due to another pathogen.
(C)     The patient was ill with swine flu on 12/20.
(D)     None of the above.

5.     A patient's serum titer was read as 80 initially and as 160 one month later in a test that used serial
twofold dilutions both times. Choose the most accurate interpretation of this data.
(A)    The amount of antibody in his serum has doubled.
(B)    The amount of antibody in his serum has increased between a few percent and almost fourfold.
(C)    The amount of antibody in his serum has increased between a few percent and 100%.
(D)    None of the above.

6.     A major problem encountered with the current influenza vaccine is:
(A)    Induction of neurological complications in about 1:10000 vaccinees
(B)    Loss of efficiency due to influenza virus drift
(C)    Poor immunogenicity of the viral polysaccharide used to prepare the vaccine
(D)    Possible infection of immunocompromised individuals
(E)    Reversion to disease-causing wild type

7.     The mechanism of action of zanamavir and oseltamavir involves:
(A)    Activation of the viral neuraminidase
(B)    Inhibition of the endonuclease that cuts the "cap" from nascent mammalian mRNA
(C)    Inhibition of the release of viral nucleocapsids into the cytoplasm of non-infected cells
(D)    Inhibition of viral propagation to non-infected cells
(E)    Interference with the viral protease




                                                                                                      58
Influenza
Questions 8 through 11 refer to the following HI pattern:




Using:

as symbol for red blood cell (RBC)


as symbol for antibody (Ab)


as symbol for virus

Match the following drawings with the wells. Each answer may only be used once.




8.   Is the 1:10 well represented by drawing a, b, c, d, or e?

9.   Is the 1:80 well represented by drawing a, b, c, d, or e?

10. Is the 1:320 well represented by drawing          a, b, c, d, or e?

11. Is the virus control represented by drawing       a, b, c, d, or e?

When you have finished the posttest, discuss your answers with your colleagues and then compare them
with the correct answers. (See last pages in the workbook, or follow your instructor’s directions.)




                                                                                              59
Influenza

Posttest Correct Answers
Discuss the answers with each other to be sure none of you have any misconceptions!

1.     A is correct. None of the wells show HI; hence, the titer is less than the reciprocal of the lowest
       serum dilution tested.

2      C is correct. The highest dilution to demonstrate activity is 1:80; hence, its reciprocal is the titer.

3      C is correct. The patient showed at least a 16-fold increase in antibody. This can be accounted for
       by either an immunization or an infection, but a history is required to determine which.

4.     B is correct. Had the first illness been swine flu, her titer would have been much higher on Nov. 30.
       Titers start to rise about one week after the onset of infection and reach a maximum two to three
       weeks after the infection.

5.     B is correct. Theoretically, the titer could have gone from 159 to 160 or from 80 to 319 and still have
       given the same results.

6.     B is correct. Although the formulation of the vaccine is updated yearly, the continuous antigenic drift
       of the virus results in loss of efficiency.

7.     D is correct. Zanamavir and oseltamavir inhibit the viral neuraminidase and, as a consequence, viral
       propagation to non-infected cells is blocked.

8.     C is correct. Antibody to the RBCs is agglutinating them (see positive serum control), and antibody
       to the virus is "coating" the virus.

9.     B is correct. The antibody to the RBC has been "diluted out," but there is still ample antibody to the
       virus to prevent it from agglutinating the RBCs. There was a higher titer of antibody to virus than to
       the RBCs.

10.    D is correct. It has some antibody (about 1/4 that shown in B), whereas E has none (E would be the
       virus control). The virus is agglutinating the RBCs.

11.    E is correct. There are only RBCs agglutinated by virus and no antibody.




When your group has finished reviewing the posttest, you have completed the activity. Have you achieved
the objectives set forth in the Introduction? Some of you may wish to discuss your reactions to this Patient-
Oriented Problem-Solving session with your instructor.


                                                                                                       60
Influenza
Posttest Correct Answers
Discuss the answers with each other to be sure none of you have any misconceptions!

1.     A is correct. None of the wells show HI; hence, the titer is less than the reciprocal of the lowest
       serum dilution tested.

2      C is correct. The highest dilution to demonstrate activity is 1:80; hence, its reciprocal is the titer.

3      C is correct. The patient showed at least a 16-fold increase in antibody. This can be accounted for
       by either an immunization or an infection, but a history is required to determine which.

4.     B is correct. Had the first illness been swine flu, her titer would have been much higher on Nov. 30.
       Titers start to rise about one week after the onset of infection and reach a maximum two to three
       weeks after the infection.

5.     B is correct. Theoretically, the titer could have gone from 159 to 160 or from 80 to 319 and still have
       given the same results.

6.     B is correct. Although the formulation of the vaccine is updated yearly, the continuous antigenic drift
       of the virus results in loss of efficiency.

7.     D is correct. Zanamavir and oseltamavir inhibit the viral neuraminidase and, as a consequence, viral
       propagation to non-infected cells is blocked.

8.     C is correct. Antibody to the RBCs is agglutinating them (see positive serum control), and antibody
       to the virus is "coating" the virus.

9.     B is correct. The antibody to the RBC has been "diluted out," but there is still ample antibody to the
       virus to prevent it from agglutinating the RBCs. There was a higher titer of antibody to virus than to
       the RBCs.

10.    D is correct. It has some antibody (about 1/4 that shown in B), whereas E has none (E would be the
       virus control). The virus is agglutinating the RBCs.

11.    E is correct. There are only RBCs agglutinated by virus and no antibody.




When your group has finished reviewing the posttest, you have completed the activity. Have you achieved
the objectives set forth in the Introduction? Some of you may wish to discuss your reactions to this Patient-
Oriented Problem-Solving session with your instructor.



                                                                                                       61
Influenza
Posttest Correct Answers
Discuss the answers with each other to be sure none of you have any misconceptions!

1.     A is correct. None of the wells show HI; hence, the titer is less than the reciprocal of the lowest
       serum dilution tested.

2      C is correct. The highest dilution to demonstrate activity is 1:80; hence, its reciprocal is the titer.

3      C is correct. The patient showed at least a 16-fold increase in antibody. This can be accounted for
       by either an immunization or an infection, but a history is required to determine which.

4.     B is correct. Had the first illness been swine flu, her titer would have been much higher on Nov. 30.
       Titers start to rise about one week after the onset of infection and reach a maximum two to three
       weeks after the infection.

5.     B is correct. Theoretically, the titer could have gone from 159 to 160 or from 80 to 319 and still have
       given the same results.

6.     B is correct. Although the formulation of the vaccine is updated yearly, the continuous antigenic drift
       of the virus results in loss of efficiency.

7.     D is correct. Zanamavir and oseltamavir inhibit the viral neuraminidase and, as a consequence, viral
       propagation to non-infected cells is blocked.

8.     C is correct. Antibody to the RBCs is agglutinating them (see positive serum control), and antibody
       to the virus is "coating" the virus.

9.     B is correct. The antibody to the RBC has been "diluted out," but there is still ample antibody to the
       virus to prevent it from agglutinating the RBCs. There was a higher titer of antibody to virus than to
       the RBCs.

10.    D is correct. It has some antibody (about 1/4 that shown in B), whereas E has none (E would be the
       virus control). The virus is agglutinating the RBCs.

11.    E is correct. There are only RBCs agglutinated by virus and no antibody.




When your group has finished reviewing the posttest, you have completed the activity. Have you achieved
the objectives set forth in the Introduction? Some of you may wish to discuss your reactions to this Patient-
Oriented Problem-Solving session with your instructor.



                                                                                                       62
Influenza
Posttest Correct Answers
Discuss the answers with each other to be sure none of you have any misconceptions!

1.     A is correct. None of the wells show HI; hence, the titer is less than the reciprocal of the lowest
       serum dilution tested.

2      C is correct. The highest dilution to demonstrate activity is 1:80; hence, its reciprocal is the titer.

3      C is correct. The patient showed at least a 16-fold increase in antibody. This can be accounted for
       by either an immunization or an infection, but a history is required to determine which.

4.     B is correct. Had the first illness been swine flu, her titer would have been much higher on Nov. 30.
       Titers start to rise about one week after the onset of infection and reach a maximum two to three
       weeks after the infection.

5.     B is correct. Theoretically, the titer could have gone from 159 to 160 or from 80 to 319 and still have
       given the same results.

6.     B is correct. Although the formulation of the vaccine is updated yearly, the continuous antigenic drift
       of the virus results in loss of efficiency.

7.     D is correct. Zanamavir and oseltamavir inhibit the viral neuraminidase and, as a consequence, viral
       propagation to non-infected cells is blocked.

8.     C is correct. Antibody to the RBCs is agglutinating them (see positive serum control), and antibody
       to the virus is "coating" the virus.

9.     B is correct. The antibody to the RBC has been "diluted out," but there is still ample antibody to the
       virus to prevent it from agglutinating the RBCs. There was a higher titer of antibody to virus than to
       the RBCs.

10.    D is correct. It has some antibody (about 1/4 that shown in B), whereas E has none (E would be the
       virus control). The virus is agglutinating the RBCs.

11.    E is correct. There are only RBCs agglutinated by virus and no antibody.




When your group has finished reviewing the posttest, you have completed the activity. Have you achieved
the objectives set forth in the Introduction? Some of you may wish to discuss your reactions to this Patient-
Oriented Problem-Solving session with your instructor.



                                                                                                       63

				
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