Recent Advances in Viral
Ondiagnosis
Jan 12 and 13, 1978, a workshop and useful methods in viral was held at the National Institutes of Health, Bethesda, Md, sponsored by the National Institute of Allergy and Infectious Disease and the North American Group for Rapid Viral Diagnosis. The workshop was designed to review recent advances in viral diagnostic techniques, a subject which should be of interest to all practicing pediatricians. The last decade has seen the development of a number of methods that have accelerated the time between the submission of a specimen by a clinician and the reception of useful information on the presence and nature of an infecting virus. Because of these advances, it is quite possible that in the next decade viral diagnosis will become inexpensive, rapid enough to be clinically useful, and widely available to clinicians in the United States. It is the purpose of this Marginal Comment to review briefly the areas in which advances have been made and to point out where there is clear promise of advances in the future. Traditional viral diagnosis has de¬ pended on analysis of acute and convalescent serums or on the culture and subsequent recognition of viruses from clinical specimens. Serologie diagnosis almost always gives a diag¬ nosis only in retrospect. Cultural tech¬ niques require the growth and main¬ tenance of tissue cultures, a technolo¬ gy considered outside the capacity of most microbiology laboratories. More¬ over, the time required to recognize virus growth, has been considered impractically long, although in our experience this assumption has fre¬ quently proved to be wrong. Most of the new techniques attempt to circum¬ vent this virus growth procedure by the detection of antigens (as opposed to live infectious viruses) in body fluids. One new method that has been clearly proved useful is that of iramunofluorescence. Professor Phillip S. Gardner of the Royal Victoria Infiron new
mary,
Diagnosis
En¬ and immunofluores-
Newcastle-upon-Tyne,
gland, has pioneered this field
demonstrated that cence could be used to identify viruses in respiratory secretions rapidly, pre¬ cisely, and with great sensitivity. The technique depends on the demonstra¬ tion of viral antigens in cells shed from the respiratory mucosa. Epithe¬ lial cells, collected by aspiration of nasal mucus, are stained for specific viral antigen using animal serums and
fluorescein-conjugated antiglobulins. Professor Gardner detects respiratory viruses
as
often
as or more
often than
they can be cultured in tissue culture, usually giving the clinician the specif¬ ic diagnosis on the same day as the specimen was obtained. The technique can be used on slides made in an outlying hospital and then sent by mail to his laboratory. He has demonstrated the feasibility of this technique in the identification of influenza virus, parainfluenza viruses, respiratory syncytial virus, adenovirus, measles, mumps, and even coronaviruses in respiratory secretions. Others have developed the technique for rubella, cytomegalovirus, varicella zoster virus, and herpes simplex. Despite this impressive record, fluo¬ rescence has never been widely prac¬ ticed for viral diagnosis within this country. The technique requires spe¬ cific antiserums of high quality, many
of which are not available commercial¬ ly at the present time. Moreover, an expensive instrument, a fluorescence microscope, must be used, and careful training of the fluorescence microscopist is an absolute necessity. These requirements tend to limit its applica¬ tion to a few academic and tertiary medical centers. There is, however, no good reason why application should not become more widespread, particu¬ larly if high quality serums become commercially available and if the benefits of such diagnosis are shown to be economically important in the of hospitalized children. As care discussed below, however, other tech¬ niques for identification of viruses in
clinical specimens may become avail¬ able that are simpler and less expen¬ sive, and fluorescence may be re¬ served for special situations. Other methods for detection of viral antigens in clinical specimens have been pioneered in those diseases where virus culture has been unsuc¬ cessful. These are, in particular, hepa¬ titis A and and viral diarrheas. In these diseases, virologists have been forced to develop strategies that do not depend on the capacity of cell culture or laboratory animals to sup¬ port viral replication. The techniques of greatest use are ones that detect very small amounts of free viral anti¬ gen in body fluids. These techniques fall into two broad categories. First, there are those that maximize the speed and sensitivity of a single antigen-antibody reaction. Examples of this are counter immunoelectrophoresis (CIE, widely used in detection of bacterial antigens), and tests where antibody is attached to the surface of larger particles, such as latex balls or erythrocytes (indirect hemagglutination tests) so that the immunologie reaction is magnified to become visible to the naked eye. The second category involves the use of two or more different antibodies, often added in a "sandwich" so that they attach to the antigen in several places or even to each other, augment¬ ing the sensitivity of the reaction at each step. Both CIE and indirect hemaggluti¬ nation have been used with success in detection of hepatitis virus, and CIE has been recently applied to fecal rotaviruses. They are practical meth¬ ods because they are simple and rapid (complete in a few hours) and require few articles of expensive equipment. They are, however, less sensitive than the second, sandwich, group of tech¬
niques.
The two most widely applied sand¬ wich techniques are radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). In both systems, one species of antiviral anti-
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body (the "capture" antibody) is attached to a solid surface, such as a plastic well or tube, and the clinical specimen is then added. The virus is allowed to attach, and then an anti¬ body or series of antibodies is added on top. The final antibody layer is labelled in some way, either with iodine 125 (for RIA), or by conjugation with an enzyme that produces a color reaction when the appropriate sub¬ strate is added (for ELISA). Radioac¬ tivity, in the former system, or color in the latter signals the presence of antigen (virus) in the specimen. Radioimmunoassay techniques
are
widely available for detection of hepatitis antigen. Enzyme-linked now
immunosorbent assays however, offer the distinct advantage that a -radioactivity counter is not necessary; the color change can be qualitatively detected by eye and quantitatively measured with a spectrophotometer,
available in most hospital chemistry laboratories. This test can also be completed quickly, usually in four to 24 hours. Enzyme-linked immunosorbent as¬ say techniques of great sensitivity have been developed for detection of hepatitis A and viruses and rotaviruses in stool. A method has been recently described for detecting herpes simplex virus in skin'vesicles. The technique holds promise for rapid and inexpensive virologie diagnosis of many other viral diseases, including respiratory illness, other gastrointes¬ tinal infections, and perhaps other herpetic conditions. As long as the antiserums and plastics used are of a high quality, the tests are almost
"goof-proof."
Considerable work still must be done to develop the full potentiality of these tests, and after this to make them practical and cheap. For some
viruses this may never happen, and we may be forced to rely on the tradition¬ al methods of tissue culture identifica¬ tion. An optimist's view is, however, that, before too long, clinicians will have available in their hospital labora¬ tories the capability to identify quick¬ ly and precisely many of the viral illnesses of childhood. If this does indeed occur, then we can hope for more rational use of antibiotics, better control of hospital-acquired infec¬ tions, and the eventual disappearance of that waste-basket diagnosis, the "viral syndrome." KENNETH MCINTOSH, MD Pediatric Infectious Disease Mail Container #C227 University of Colorado Medical Center 4200 E Ninth Ave Denver, CO 80220
Use of Modified Food Starches in Infant Nutrition foodstarches (MFS) are food additives used to impart functional properties to food products. Modified food starches are used primarily in strained and junior foods and to a minor extent in infant formulas, such as soybean (Isomil). Recently, the American Academy of Pediatrics subcommittee, under contract to the Food and Drug Administration, held a conference on the use and safety of MFSs in infant nutrition. (The subcommittee on evaluation of safety of modified starches in infant foods, committee on nutrition, American
Modified
Academy
of
Pediatrics.1) This compart on the subcom-
ment is based in
mittee's recommendations. Interest in the MFS arises from three principal concerns. The first relates to the bioavailability of the starch itself. The second is the potential that undigestible starch may have for producing diarrheal symptoms, malabsorption, and changes in gastrointestinal (GI) flora. The third is the toxicological effect of the chemicals used to modify the starch and their possible mutagenic and carcin-
ogenic properties.
There have been few long-term studies to delineate the effect of starch feeding on the growth of young infants. Nonetheless, during the past half a century, there has been an increasing tendency to introduce solid foods (beikost) to infants very early in life, and many infants receive cereal and other carbohydrate-containing foods as early as the first, second, and third weeks of life. As a result, abnor¬ mally loose stools are not uncommon in these infants. Is this deleterious to the infant? The answer to this impor¬ tant question is not known.
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/26/2015
Ondiagnosis
Jan 12 and 13, 1978, a workshop and useful methods in viral was held at the National Institutes of Health, Bethesda, Md, sponsored by the National Institute of Allergy and Infectious Disease and the North American Group for Rapid Viral Diagnosis. The workshop was designed to review recent advances in viral diagnostic techniques, a subject which should be of interest to all practicing pediatricians. The last decade has seen the development of a number of methods that have accelerated the time between the submission of a specimen by a clinician and the reception of useful information on the presence and nature of an infecting virus. Because of these advances, it is quite possible that in the next decade viral diagnosis will become inexpensive, rapid enough to be clinically useful, and widely available to clinicians in the United States. It is the purpose of this Marginal Comment to review briefly the areas in which advances have been made and to point out where there is clear promise of advances in the future. Traditional viral diagnosis has de¬ pended on analysis of acute and convalescent serums or on the culture and subsequent recognition of viruses from clinical specimens. Serologie diagnosis almost always gives a diag¬ nosis only in retrospect. Cultural tech¬ niques require the growth and main¬ tenance of tissue cultures, a technolo¬ gy considered outside the capacity of most microbiology laboratories. More¬ over, the time required to recognize virus growth, has been considered impractically long, although in our experience this assumption has fre¬ quently proved to be wrong. Most of the new techniques attempt to circum¬ vent this virus growth procedure by the detection of antigens (as opposed to live infectious viruses) in body fluids. One new method that has been clearly proved useful is that of iramunofluorescence. Professor Phillip S. Gardner of the Royal Victoria Infiron new
mary,
Diagnosis
En¬ and immunofluores-
Newcastle-upon-Tyne,
gland, has pioneered this field
demonstrated that cence could be used to identify viruses in respiratory secretions rapidly, pre¬ cisely, and with great sensitivity. The technique depends on the demonstra¬ tion of viral antigens in cells shed from the respiratory mucosa. Epithe¬ lial cells, collected by aspiration of nasal mucus, are stained for specific viral antigen using animal serums and
fluorescein-conjugated antiglobulins. Professor Gardner detects respiratory viruses
as
often
as or more
often than
they can be cultured in tissue culture, usually giving the clinician the specif¬ ic diagnosis on the same day as the specimen was obtained. The technique can be used on slides made in an outlying hospital and then sent by mail to his laboratory. He has demonstrated the feasibility of this technique in the identification of influenza virus, parainfluenza viruses, respiratory syncytial virus, adenovirus, measles, mumps, and even coronaviruses in respiratory secretions. Others have developed the technique for rubella, cytomegalovirus, varicella zoster virus, and herpes simplex. Despite this impressive record, fluo¬ rescence has never been widely prac¬ ticed for viral diagnosis within this country. The technique requires spe¬ cific antiserums of high quality, many
of which are not available commercial¬ ly at the present time. Moreover, an expensive instrument, a fluorescence microscope, must be used, and careful training of the fluorescence microscopist is an absolute necessity. These requirements tend to limit its applica¬ tion to a few academic and tertiary medical centers. There is, however, no good reason why application should not become more widespread, particu¬ larly if high quality serums become commercially available and if the benefits of such diagnosis are shown to be economically important in the of hospitalized children. As care discussed below, however, other tech¬ niques for identification of viruses in
clinical specimens may become avail¬ able that are simpler and less expen¬ sive, and fluorescence may be re¬ served for special situations. Other methods for detection of viral antigens in clinical specimens have been pioneered in those diseases where virus culture has been unsuc¬ cessful. These are, in particular, hepa¬ titis A and and viral diarrheas. In these diseases, virologists have been forced to develop strategies that do not depend on the capacity of cell culture or laboratory animals to sup¬ port viral replication. The techniques of greatest use are ones that detect very small amounts of free viral anti¬ gen in body fluids. These techniques fall into two broad categories. First, there are those that maximize the speed and sensitivity of a single antigen-antibody reaction. Examples of this are counter immunoelectrophoresis (CIE, widely used in detection of bacterial antigens), and tests where antibody is attached to the surface of larger particles, such as latex balls or erythrocytes (indirect hemagglutination tests) so that the immunologie reaction is magnified to become visible to the naked eye. The second category involves the use of two or more different antibodies, often added in a "sandwich" so that they attach to the antigen in several places or even to each other, augment¬ ing the sensitivity of the reaction at each step. Both CIE and indirect hemaggluti¬ nation have been used with success in detection of hepatitis virus, and CIE has been recently applied to fecal rotaviruses. They are practical meth¬ ods because they are simple and rapid (complete in a few hours) and require few articles of expensive equipment. They are, however, less sensitive than the second, sandwich, group of tech¬
niques.
The two most widely applied sand¬ wich techniques are radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). In both systems, one species of antiviral anti-
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/26/2015
body (the "capture" antibody) is attached to a solid surface, such as a plastic well or tube, and the clinical specimen is then added. The virus is allowed to attach, and then an anti¬ body or series of antibodies is added on top. The final antibody layer is labelled in some way, either with iodine 125 (for RIA), or by conjugation with an enzyme that produces a color reaction when the appropriate sub¬ strate is added (for ELISA). Radioac¬ tivity, in the former system, or color in the latter signals the presence of antigen (virus) in the specimen. Radioimmunoassay techniques
are
widely available for detection of hepatitis antigen. Enzyme-linked now
immunosorbent assays however, offer the distinct advantage that a -radioactivity counter is not necessary; the color change can be qualitatively detected by eye and quantitatively measured with a spectrophotometer,
available in most hospital chemistry laboratories. This test can also be completed quickly, usually in four to 24 hours. Enzyme-linked immunosorbent as¬ say techniques of great sensitivity have been developed for detection of hepatitis A and viruses and rotaviruses in stool. A method has been recently described for detecting herpes simplex virus in skin'vesicles. The technique holds promise for rapid and inexpensive virologie diagnosis of many other viral diseases, including respiratory illness, other gastrointes¬ tinal infections, and perhaps other herpetic conditions. As long as the antiserums and plastics used are of a high quality, the tests are almost
"goof-proof."
Considerable work still must be done to develop the full potentiality of these tests, and after this to make them practical and cheap. For some
viruses this may never happen, and we may be forced to rely on the tradition¬ al methods of tissue culture identifica¬ tion. An optimist's view is, however, that, before too long, clinicians will have available in their hospital labora¬ tories the capability to identify quick¬ ly and precisely many of the viral illnesses of childhood. If this does indeed occur, then we can hope for more rational use of antibiotics, better control of hospital-acquired infec¬ tions, and the eventual disappearance of that waste-basket diagnosis, the "viral syndrome." KENNETH MCINTOSH, MD Pediatric Infectious Disease Mail Container #C227 University of Colorado Medical Center 4200 E Ninth Ave Denver, CO 80220
Use of Modified Food Starches in Infant Nutrition foodstarches (MFS) are food additives used to impart functional properties to food products. Modified food starches are used primarily in strained and junior foods and to a minor extent in infant formulas, such as soybean (Isomil). Recently, the American Academy of Pediatrics subcommittee, under contract to the Food and Drug Administration, held a conference on the use and safety of MFSs in infant nutrition. (The subcommittee on evaluation of safety of modified starches in infant foods, committee on nutrition, American
Modified
Academy
of
Pediatrics.1) This compart on the subcom-
ment is based in
mittee's recommendations. Interest in the MFS arises from three principal concerns. The first relates to the bioavailability of the starch itself. The second is the potential that undigestible starch may have for producing diarrheal symptoms, malabsorption, and changes in gastrointestinal (GI) flora. The third is the toxicological effect of the chemicals used to modify the starch and their possible mutagenic and carcin-
ogenic properties.
There have been few long-term studies to delineate the effect of starch feeding on the growth of young infants. Nonetheless, during the past half a century, there has been an increasing tendency to introduce solid foods (beikost) to infants very early in life, and many infants receive cereal and other carbohydrate-containing foods as early as the first, second, and third weeks of life. As a result, abnor¬ mally loose stools are not uncommon in these infants. Is this deleterious to the infant? The answer to this impor¬ tant question is not known.
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/26/2015
