Table Of ContentThis book is dedicated to the
memory of Sarah Gardner
Rapid virus diagnosis
Application ofimmunofluorescence
Second edition
P. S. GARDNER, MD, DipBact
Formerly Professor of Clinical Virology, Royal
Victoria Infirmary and University of
Newcastle upon Tyne; Director, Microbiological Reagents
and Quality Control, Central Public Health
Laboratory, Colindale, London
and
J. McQUILLIN, BSc, FIMLS
Principal Scientific Officer and Honorary
Lecturer in Clinical Virology, Royal Victoria
Infirmary and University of Newcastle upon Tyne
Assisted by
M. Grandien, MD
Associate Professor of Virology,
National Bacteriological Laboratory,
Stockholm, Sweden
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First published 1974
Second edition 1980
© Butterworth & Co (Publishers) Ltd 1980
ISBN 0 407 38441 3
British Library Cataloguing
in Publication Data
Gardner, Phillip Samuel
Rapid virus diagnosis. - 2nded.
1. Virus diseases - Diagnosis
2. Immunofluorescence
I. Title II. McQuillin, Joyce III. Grandien, M
616.9'2'07572 RC114 80-40239
ISBN 0407 384413
Printed in Great Britain by
The Whitefriars Press Ltd., London and Tonbridge
Foreword
It would be wrong to suppose that the privilege of being asked to write this
foreword is in any way the result of past sponsorship by me of diagnosis by
the fluorescent antibody technique. Until I had read this book, I was no
such advocate, but merely a devotee of this most seductive method of
following viruses around. Ignorance of the method's diagnostic
effectiveness and lack of a comprehensive, critical assessment of its uses
over a wide range of virus infections have been the chief deterrents to its
widespread application.
Now Professor Gardner and Miss McQuillin have provided an estimable
remedy for this state of affairs. In these pages their own wide experience
and the published results of other reliable investigators in the same field are
so combined that, without false encouragement, the reader finds himself
able to satisfy the four chief needs of laboratory diagnosis—where and
when to apply the test, how exactly to do it, what results to expect and how
to draw safe conclusions.
All those who take their first steps in diagnostic virology and all those
who wish to further their skills will be glad, as I shall, to have this book.
K. B. Fraser
Department of Microbiology,
The Queen's University of Belfast
V
Preface to second edition
The view expressed in the Preface to the first edition that immuno-
fluorescence as a technique for the diagnosis of virus infections was
becoming increasingly used by virologists has been fully justified. As with
any biological system, even over the space of a few years, there have been
changes and new ideas and developments need to be incorporated. This
has therefore necessitated the production of a second edition. Over the
intervening 4-5 years we have been delighted to see the increasing interest
that clinicians have taken in our efforts; this interest has helped to form the
partnership that now exists between them and laboratory workers. The
intervening years have also seen the worldwide growth of interest in rapid
virus diagnostic techniques. The European Group for Rapid Laboratory
Viral Diagnosis was formed in 1975, the World Health Organization has
an increasing involvement in rapid virus diagnosis and in 1977 the Pan
American Group for Rapid Laboratory Viral Diagnosis was formed.
While we fully recognize that the fluorescent antibody is one of a number
of techniques, all with their own uses, it nevertheless forms the foundation
on which the subject was built and on which it is still expanding.
Much of the material of the first edition has naturally been kept and our
own thanks go again to those we acknowledged in our previous edition for
use of photographs and figures. We also again freely refer to Fluorescent
Protein Tracing by Nairn (1976) and we hope that the reader will find our
own monograph complementary in the virus field to that admirable
textbook.
To this new edition we have added two chapters: the first was one
designed to acknowledge the use of IgM detection in rapid virus diagnosis
and the second to emphasize the many applications of rapid virus
diagnosis in the clinical field.
We were fortunate to receive the assistance of Dr Monica Grandien of
Stockholm in this new edition and we are especially grateful for her
contribution to the chapter on rabies and for the new chapter on detection
of virus-specific IgM. We must also express our gratitude to all members of
the laboratory staff of the Virus Department at Newcastle upon Tyne,
especially Miss R. McGuckin, SCMLSO, for their continued help;
secretarial help has been given by Miss C. Montgomery (University
Vll
viii Preface to second edition
Secretary) and Mrs R. Frazer (Hospital Department Secretary) for which
we express our thanks.
Perhaps the sincerest thanks of all should go to our long-suffering
publishers, Butterworths, who have maintained a patience and under-
standing which is outside the normal call of duty.
It is with great regret that we record the death of Miss Sarah Gardner, a
most courageous young lady, who designed the cover of the original
edition. It would seem appropriate to dedicate this second edition to her
memory.
Finally we hope that the reader of this new edition finds it a useful
addition to his laboratory shelf and that the new information added will
make immunofluorescence glow even brighter in the virological diagnostic
field.
P. S. Gardner and Joyce McQuillin
Preface to first edition
It has become clear over the last few years that immunofluorescence as a
technique for the diagnosis of virus infections is becoming increasingly
used by virologists. Consequently, we consider that the time is now ripe to
collect together as a monograph the techniques that may be used and the
virus infections to which these techniques may be applied. In this way, we
hope that this book will prove useful, not only to virologists both medical
and non-medical, but to technicians applying these techniques and to
physicians who wish to know which infections may be investigated in this
way. We feel that virus diagnosis is a partnership between virologists,
technicians, and clinicians; all should know the material required and the
method for their transport to the laboratory so that optimal results may be
obtained.
We have tried, as far as possible, to base our work on personal
experience, but in areas where our knowledge has been less extensive, we
have been fortunate enough to be able to use material from colleagues; in
particular we wish to record our thanks to Professor K. B. Fraser,
Dr Margaret Haire, Dr J. Banatvala and Dr T. Bell, who have allowed
us to use photographs of their results.
We would also note here our admiration for two textbooks to which we
have made constant reference throughout our monograph: Fluorescent
Antibody Methods, by Goldman, M. (1968), and Fluorescent Protein
Tracing, by Nairn, R. C. (1969). Our own effort in no way rivals these
works but is intended to complement them in the virus field.
As much of our information is based on our own laboratory material, all
our laboratory staff have contributed to this work and our thanks must go
to all the technicians, particularly Miss R. McGuckin, Senior Chief
Technician, the Graduate staff, and to Mrs E. A. Mackay, the
Departmental Secretary.
We must also note the help we have received in producing a number of
our figures from Wild (Heerbrugg) UK Limited, and Vickers (Instruments)
Limited.
Our gratitude must also be expressed to Sterling-Winthrop Research
and Development whose original conjugate, so generously given, set us on
this long and colourful journey, and we must also thank our publishers
ix
x Preface to first edition
Butterworths who have shown patience and understanding during the
development of this work.
We are also grateful to Miss Sarah Gardner who gave freely of her time
and originality to design the book cover.
Finally, we hope that you the reader will find this book an indispensable
bench companion and that our own experiences will spare you some of the
trials and tribulations that we have undergone.
P. S. Gardner and Joyce McQuillin
1974
1
Introduction
Although many scientific discoveries have marked the progress of
medicine, these successes are still ultimately measured in terms of the
benefit derived by the patient. These benefits to the patient are usually
expressed in terms of successful management and treatment which, in turn,
depend on diagnosis. The precise diagnosis of an illness has therefore
become an integral part of treatment and the more speedily and accurately
the aetiological cause of an illness is made known, the sooner will rational
therapy be instituted. This point is well illustrated by the application of
chemotherapy to bacterial infections. Virus infections are no exception to
this general rule but specific antiviral therapy is still at a very early stage of
development and only few drugs are available at present. Methisazone has
been used in prophylaxis and treatment of poxvirus infections and
idoxuridine is effective against herpesvirus hominis. Cytarabine and
vidarabine also have an effect on the herpesvirus group. Amantadine
hydrochloride and the isoquinolines have been shown to produce a
prophylactic effect when volunteers and patients are exposed to influenza
and some believe they have some therapeutic effect as well. Interferon too
has been shown to have an antiviral effect in vivo. It is hoped that these are
the forerunners of many more antiviral drugs. Much of the apathy in
research on antiviral agents and treatment of virus diseases has been due to
the time-consuming methods employed for the identification of the causal
viruses; the time taken for their identification may vary from one week to a
month or even longer. The need was apparent for development of effective
and rapid methods for the diagnosis of virus infections. Rapid diagnosis of
a virus infection is a relative term but in this context should be interpreted
in hours.
This book describes the application of fluorescent antibody techniques
to the diagnosis and the investigation of virus infections and the assessment
of their value both to the clinician and to the virologist but fully recognizes
that other rapid techniques have an important and developing role. Some
techniques are more suited for detecting virus in particular situations and
up to the present the method of choice for the rapid diagnosis of acute
virus infections in the respiratory tract is immunofluorescence. Electron
microscopy is the method of choice for the diagnosis of certain virus
1
2 Introduction
infections as exemplified by the poxviruses, wartvirus and orf. On other
occasions a number of methods can be used: electron microscopy and the
fluorescent antibody technique for the detection of herpesvirus infections of
superficial lesions and the brain; enzyme-linked immunosorbent assay
(ELISA) and electron microscopy for rotavirus infections. The choice here
usually depends on the expertise present in a department and also on the
apparatus available. Rapid virus diagnosis has become the 'raison d'être*
of clinical virology and organizations such as the European Group for
Rapid Laboratory Viral Diagnosis and the Pan American Group for Rapid
Laboratory Virus Diagnosis have evolved to assist virologists in obtaining
suitable reagents and developing suitable technology. These two
organizations have with the assistance of the World Health Organization
initiated a number of training schemes in these techniques together with a
number of symposia. The World Health Organization itself has a great
interest in these developments as some of the techniques may bring a
measure of virus diagnosis to developing countries handicapped by limited
economic and technical resources. The subject has been reviewed recently
(Gardner, 1977; Mclntosh, 1978). This book attempts to indicate to the
reader the method of choice for each virus group and the relative role that
immunofluorescence can play in clinical virology.
There are few comprehensive up-to-date textbooks on immuno-
fluorescence; constant reference will be made to Nairn (1976) though
a number of general textbooks are now recognizing immunofluorescence as
a diagnostic tool (Emmons and Riggs, 1977; Lennette and Schmidt, 1979;
Lyerla, 1979; Nakamura, Dito and Tucker, 1979), but the importance of
the application of immunofluorescence through rapid virus identification
has still to be put into perspective. Acknowledgement must be made to
earlier books on the subject to which reference can still be made with
purpose (Goldman, 1968; Holborow, 1970; Beutner, 1971). The addition
of another book to the already overcrowded shelf of the doctor, student,
scientist or technician should not be undertaken lightly unless immuno-
fluorescent diagnosis can be shown to have a unique place in
diagnostic virology. This we believe to be the case. Many virus infections
produce their lesions in accessible sites or are present in secretions from
these sites, which facilitates the taking of specimens for investigation. Skin
and corneal scrapings in infections with herpesvirus hominis and secretions
taken from the nasopharynx in respiratory infections are examples of this.
Modern biopsy methods, too, allow an ever-increasing source of material
for the rapid diagnosis of virus infection from sites until recently
considered inaccessible. An example of this is brain biopsy for suspected
herpesvirus hominis encephalitis. Therefore, if the virus is present in an
infection and material can be obtained which contains viral antigen, there
is no theoretical reason why a fluorescent antibody technique cannot be
employed to diagnose its presence in a few hours. There are other reasons,
however, which may make it impractical to diagnose a disease by
