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Rapid laboratory diagnosis of respiratory viral infections
Journal of Infection (1982) 5, 241-243
LEADING ARTICLE
Rapid l a b o r a t o r y diagnosis o f respiratory viral infections J. Gray Public Health Laboratory, Central Pathology Laboratory, Hartshill Road, Hartshill, Stoke-on-Trent
Respiratory viral infections Acute respiratory infections are a major cause of morbidity all over the world. M a n y of these infections are caused by viruses. Some such as the common cold are mild diseases whereas others such as measles and influenza may be severe illnesses. In very young children the major pathogen is respiratory syncytial virus. Classical laboratory techniques used in diagnostic virology are slow, expensive and may be difficult or impossible to perform in all laboratories. In recent years several techniques for rapid virus diagnosis have been developed which have other benefits apart from the obvious one of speed. 1, 2. a Some viruses cannot be cultivated in conventional tissue culture systems and can only be detected by immunological methods. Other viruses which do grow in tissue culture can be identified more quickly using immunological techniques. Antiviral drugs have become available and although their use is still very limited the further development of them necessitates accurate rapid diagnosis. In hospitals the rapid identification of infectious patients allows measures to be taken to reduce the risk of cross-infection. Conventional virological techniques for the diagnosis of respiratory infections are based on the isolation of the virus or antibody detection. Antibodies to respiratory viruses are not present at the time of the acute infection, so rapid methods to detect antibodies are of no value in the clinical management of a patient. Useful techniques have all been aimed at the rapid detection of viral antigens in clinical specimens. T h e fluorescent antibody technique (FAT) is the method of choice. T h e technique is well known and has been evaluated in clinical use. 4 F A T is sensitive, specific and relatively inexpensive. However, the selection of specimens is very important. T h e specimens must contain intact cells with viral antigen. T h e best specimens are nasopharyngeal secretions collected by aspiration although nasal and throat washings may give satisfactory results. It is important that mucus and pus adhering to the cells are washed away otherwise non-specific fluorescence or false negative results may be obtained. T h e cells are fixed on glass slides and can be posted to a suitable laboratory for examination if facilities are not available on site. T h e technique can be used for respiratory syncytial virus, influenza virus A and B, measles, the adenovirus group and the para-influenza viruses I, 2, 3, 4 A and 4B. Either the direct or indirect fluorescent test can be used, but if several viruses are being sought it is more convenient to use one fluorescent o163-4453/82/o6o241 +03 $02.00/0
© 1982 The British Society for the Study of Infection
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J. GRAY
conjugate and the indirect method. Highly specific antisera and good conjugates are needed. Immunofluorescence is a sensitive test but has several practical limitations. Clinicians must be aware of the service available if they are to make efficient use of it. T h e difficulty of teaching ward staff to collect nasopharyngeal secretions and the vagaries of hospital transport systems often make it necessary for a member of the laboratory staff to collect the specimen from the patient. This may be satisfactory if the patient is not far from the laboratory but is still time consuming and makes an orderly work pattern difficult. If the patient is at a distance from the laboratory such a service may be impossible. T h e method also requires a highly skilled microscopist, with a good-quality microscope, and depends on subjective assessment of the degree of fluorescence. Large numbers of samples cannot be processed readily because fluorescent antibody techniques are not easily automated. T h e range of antigens that can be tested is also limited by the supply of suitable reagents. Many are not commercially available and most laboratories do not have the facilities to produce their own or the necessary expertise to ensure the quality of them. Radioimmunoassay and enzyme immunoassay have been developed for the large-scale screening of populations for the presence of hepatitis antigen or antibody. These techniques can be used for respiratory infections. Radioimmunoassays are less popular than formerly and most development has been with the Elisa technique. This is similar in design to solid-phase radioimmunoassay but an enzyme is used instead of the radioactive isotope as the identifying marker. Enzyme systems can detect the binding of very small amounts of antigen. T h e substrate produces a visible colour which can be read by eye. This is useful when no elaborate equipment is available but in order to increase the sensitivity of the test and to get quantitative results a spectrophotometer is required. Advantages of the technique are that the reagents are relatively stable and large numbers of samples may be examined at the same time. However the procedure is relatively complicated, there are often many intermediate steps and it is time consuming. T h e plastic plates used may have an uneven coating of antibody because of the static effects induced during production. The chromogenic substrates may also be carcinogenic or mutagenic. Several groups of workers have used Elisa techniques for the detection of RSV antigen. Two groups used similar sandwich techniques, ~, Gwhile a third group used an Elisa inhibition technique. 7 All three groups found these tests to be as sensitive as F A T , but the determination of the cutoff point for negative results is of crucial importance for the sensitivity and specificity of the assay. At present the immunological methods are not as sensitive as tissue culture systems and so cannot replace them completely. Viruses with many distinct serotypes are also unsuitable because of the large number of antisera needed. For infections such as influenza, replicating virus is needed for antigenic analysis. Another problem is the supply of standardised antisera. Cloning techniques should allow the future production of large amounts of antibody to defined antigens. T h e current limitations and future prospects of immunoassay have been reviewed. 8 One way of avoiding problems of restrictive
Rapid Diagnosis of Respiratory Infections
243
i m m u n o l o g i c a l s p e c i f i c i t y a n d l o w a v i d i t y is to m e a s u r e a m i c r o b i a l e n z y m e d i r e c t l y s u c h as t h e n e u r a m i n i d a s e o f i n f l u e n z a A v i r u s ? T h e t e c h n i q u e s f o r r a p i d v i r u s d i a g n o s i s are e v o l v i n g r a p i d l y . I t is i m p o s s i b l e to tell w h i c h t e s t s will u l t i m a t e l y p r o v e m o s t useful. T h e W H O r e c o g n i s e s t h a t r a p i d m e t h o d s c a n b e u s e d in areas l a c k i n g c o n v e n t i o n a l facilities. T h e y c o u l d aid t h e p l a n n i n g a n d m o n i t o r i n g o f v a c c i n a t i o n p r o g r a m m e s . T o e n c o u r a g e this a i m W H O r u n s t r a i n i n g p r o g r a m m e s a n d u n d e r t a k e s to s u p p l y s u i t a b l e r e a g e n t s u n t i l local v i r u s l a b o r a t o r i e s are self-sufficient. ~0 T h e f u t u r e p r o s p e c t s are e x c i t i n g , b u t t h e r e is a l o n g w a y to go f r o m t h e r e s u l t s a c h i e v e d in a c a d e m i c l a b o r a t o r i e s to t h e W H O ideal o f local d i a g n o s t i c l a b o r a t o r i e s t h r o u g h o u t t h e world.
References I. Mclntosh K, Wilfert C, Chernesky M, Plotkin S, Mattheis MJ. Summary of a workshop in new and useful techniques in rapid viral diagnosis. J Infect Dis 1978; I38:414-419 • 2. McIntosh K, Wilfert C, Chernesky M, Plotkin S, Mattheis MJ. Summary of a workshop in new and useful techniques in rapid viral diagnosis. J Infect Dis 198o: I42: 793-8o2. 3. Rapid laboratory techniques for the diagnosis of viral infections. Geneva World Health Organisation Technical Report Series 661, 1981. 4. Gardner PS, McQuillin J. Rapid virus diagnosis - application of immunofluorescence, 2nd ed. London: Butterworth, 198o. 5. Chao RK, Fishaut M, Schwartzman JD, McIntosh K. Detection of respiratory syncytial virus in nasal secretions from infants by enzyme-linked immunosorbent assay. J. Infect Dis 1979; 139: 483-486. 6. Sarkkinen HK, Halonen PE, Arstila PP, Salmi AA. Detection of respiratory syncytial, parainfluenza type 2 and adenovirus antigens by radioimmunoassay and enzyme immunoassay on nasopharyngeal secretions from children with acute respiratory disease. J Clin Microbiol ~98I; 13: 258-265. 7. Hornsleth A, Brenoe E, Friis B, Knudsden FU, UldaU P. Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay. J Clin Microbiol 1981; 14: 5IO-515. 8. Yolken RH. Enzyme immunoassay for the detection of infective antigens in body fuids: current limitations and future prospects. Rev Infect Dis 1982; 4:35 -68. 9. Yolken RH, Torsch VM, Berg R, Murphy BR, Lee YC. Fluorimetric assay for the measurement of viral neuraminidase - application to the rapid detection of influenza virus in nasal wash specimens. J Infect Dis 198o; 142:516-523 • IO. Manual for rapid laboratory viral diagnosis. WHO Offset Publication No 47. Geneva: World Health Organisation, I979.
LEADING ARTICLE
Rapid l a b o r a t o r y diagnosis o f respiratory viral infections J. Gray Public Health Laboratory, Central Pathology Laboratory, Hartshill Road, Hartshill, Stoke-on-Trent
Respiratory viral infections Acute respiratory infections are a major cause of morbidity all over the world. M a n y of these infections are caused by viruses. Some such as the common cold are mild diseases whereas others such as measles and influenza may be severe illnesses. In very young children the major pathogen is respiratory syncytial virus. Classical laboratory techniques used in diagnostic virology are slow, expensive and may be difficult or impossible to perform in all laboratories. In recent years several techniques for rapid virus diagnosis have been developed which have other benefits apart from the obvious one of speed. 1, 2. a Some viruses cannot be cultivated in conventional tissue culture systems and can only be detected by immunological methods. Other viruses which do grow in tissue culture can be identified more quickly using immunological techniques. Antiviral drugs have become available and although their use is still very limited the further development of them necessitates accurate rapid diagnosis. In hospitals the rapid identification of infectious patients allows measures to be taken to reduce the risk of cross-infection. Conventional virological techniques for the diagnosis of respiratory infections are based on the isolation of the virus or antibody detection. Antibodies to respiratory viruses are not present at the time of the acute infection, so rapid methods to detect antibodies are of no value in the clinical management of a patient. Useful techniques have all been aimed at the rapid detection of viral antigens in clinical specimens. T h e fluorescent antibody technique (FAT) is the method of choice. T h e technique is well known and has been evaluated in clinical use. 4 F A T is sensitive, specific and relatively inexpensive. However, the selection of specimens is very important. T h e specimens must contain intact cells with viral antigen. T h e best specimens are nasopharyngeal secretions collected by aspiration although nasal and throat washings may give satisfactory results. It is important that mucus and pus adhering to the cells are washed away otherwise non-specific fluorescence or false negative results may be obtained. T h e cells are fixed on glass slides and can be posted to a suitable laboratory for examination if facilities are not available on site. T h e technique can be used for respiratory syncytial virus, influenza virus A and B, measles, the adenovirus group and the para-influenza viruses I, 2, 3, 4 A and 4B. Either the direct or indirect fluorescent test can be used, but if several viruses are being sought it is more convenient to use one fluorescent o163-4453/82/o6o241 +03 $02.00/0
© 1982 The British Society for the Study of Infection
242
J. GRAY
conjugate and the indirect method. Highly specific antisera and good conjugates are needed. Immunofluorescence is a sensitive test but has several practical limitations. Clinicians must be aware of the service available if they are to make efficient use of it. T h e difficulty of teaching ward staff to collect nasopharyngeal secretions and the vagaries of hospital transport systems often make it necessary for a member of the laboratory staff to collect the specimen from the patient. This may be satisfactory if the patient is not far from the laboratory but is still time consuming and makes an orderly work pattern difficult. If the patient is at a distance from the laboratory such a service may be impossible. T h e method also requires a highly skilled microscopist, with a good-quality microscope, and depends on subjective assessment of the degree of fluorescence. Large numbers of samples cannot be processed readily because fluorescent antibody techniques are not easily automated. T h e range of antigens that can be tested is also limited by the supply of suitable reagents. Many are not commercially available and most laboratories do not have the facilities to produce their own or the necessary expertise to ensure the quality of them. Radioimmunoassay and enzyme immunoassay have been developed for the large-scale screening of populations for the presence of hepatitis antigen or antibody. These techniques can be used for respiratory infections. Radioimmunoassays are less popular than formerly and most development has been with the Elisa technique. This is similar in design to solid-phase radioimmunoassay but an enzyme is used instead of the radioactive isotope as the identifying marker. Enzyme systems can detect the binding of very small amounts of antigen. T h e substrate produces a visible colour which can be read by eye. This is useful when no elaborate equipment is available but in order to increase the sensitivity of the test and to get quantitative results a spectrophotometer is required. Advantages of the technique are that the reagents are relatively stable and large numbers of samples may be examined at the same time. However the procedure is relatively complicated, there are often many intermediate steps and it is time consuming. T h e plastic plates used may have an uneven coating of antibody because of the static effects induced during production. The chromogenic substrates may also be carcinogenic or mutagenic. Several groups of workers have used Elisa techniques for the detection of RSV antigen. Two groups used similar sandwich techniques, ~, Gwhile a third group used an Elisa inhibition technique. 7 All three groups found these tests to be as sensitive as F A T , but the determination of the cutoff point for negative results is of crucial importance for the sensitivity and specificity of the assay. At present the immunological methods are not as sensitive as tissue culture systems and so cannot replace them completely. Viruses with many distinct serotypes are also unsuitable because of the large number of antisera needed. For infections such as influenza, replicating virus is needed for antigenic analysis. Another problem is the supply of standardised antisera. Cloning techniques should allow the future production of large amounts of antibody to defined antigens. T h e current limitations and future prospects of immunoassay have been reviewed. 8 One way of avoiding problems of restrictive
Rapid Diagnosis of Respiratory Infections
243
i m m u n o l o g i c a l s p e c i f i c i t y a n d l o w a v i d i t y is to m e a s u r e a m i c r o b i a l e n z y m e d i r e c t l y s u c h as t h e n e u r a m i n i d a s e o f i n f l u e n z a A v i r u s ? T h e t e c h n i q u e s f o r r a p i d v i r u s d i a g n o s i s are e v o l v i n g r a p i d l y . I t is i m p o s s i b l e to tell w h i c h t e s t s will u l t i m a t e l y p r o v e m o s t useful. T h e W H O r e c o g n i s e s t h a t r a p i d m e t h o d s c a n b e u s e d in areas l a c k i n g c o n v e n t i o n a l facilities. T h e y c o u l d aid t h e p l a n n i n g a n d m o n i t o r i n g o f v a c c i n a t i o n p r o g r a m m e s . T o e n c o u r a g e this a i m W H O r u n s t r a i n i n g p r o g r a m m e s a n d u n d e r t a k e s to s u p p l y s u i t a b l e r e a g e n t s u n t i l local v i r u s l a b o r a t o r i e s are self-sufficient. ~0 T h e f u t u r e p r o s p e c t s are e x c i t i n g , b u t t h e r e is a l o n g w a y to go f r o m t h e r e s u l t s a c h i e v e d in a c a d e m i c l a b o r a t o r i e s to t h e W H O ideal o f local d i a g n o s t i c l a b o r a t o r i e s t h r o u g h o u t t h e world.
References I. Mclntosh K, Wilfert C, Chernesky M, Plotkin S, Mattheis MJ. Summary of a workshop in new and useful techniques in rapid viral diagnosis. J Infect Dis 1978; I38:414-419 • 2. McIntosh K, Wilfert C, Chernesky M, Plotkin S, Mattheis MJ. Summary of a workshop in new and useful techniques in rapid viral diagnosis. J Infect Dis 198o: I42: 793-8o2. 3. Rapid laboratory techniques for the diagnosis of viral infections. Geneva World Health Organisation Technical Report Series 661, 1981. 4. Gardner PS, McQuillin J. Rapid virus diagnosis - application of immunofluorescence, 2nd ed. London: Butterworth, 198o. 5. Chao RK, Fishaut M, Schwartzman JD, McIntosh K. Detection of respiratory syncytial virus in nasal secretions from infants by enzyme-linked immunosorbent assay. J. Infect Dis 1979; 139: 483-486. 6. Sarkkinen HK, Halonen PE, Arstila PP, Salmi AA. Detection of respiratory syncytial, parainfluenza type 2 and adenovirus antigens by radioimmunoassay and enzyme immunoassay on nasopharyngeal secretions from children with acute respiratory disease. J Clin Microbiol ~98I; 13: 258-265. 7. Hornsleth A, Brenoe E, Friis B, Knudsden FU, UldaU P. Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay. J Clin Microbiol 1981; 14: 5IO-515. 8. Yolken RH. Enzyme immunoassay for the detection of infective antigens in body fuids: current limitations and future prospects. Rev Infect Dis 1982; 4:35 -68. 9. Yolken RH, Torsch VM, Berg R, Murphy BR, Lee YC. Fluorimetric assay for the measurement of viral neuraminidase - application to the rapid detection of influenza virus in nasal wash specimens. J Infect Dis 198o; 142:516-523 • IO. Manual for rapid laboratory viral diagnosis. WHO Offset Publication No 47. Geneva: World Health Organisation, I979.