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New techniques in rapid viral diagnosis
FEMS Microbiology Immunology 89 (1992) 299-304 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier
299
FEMSIM 00221
Review
New techniques in rapid viral diagnosis Thomas Krech In der Fina 21 a, Schaan, Liechtenstein
Received 15 March 1992 Revision received and accepted 21 April 1992
Key words: Rapid viral diagnosis; Biosensor; Nucleic acid amplification; Polymerase chain reaction; lmmunoassay
1. SUMMARY
2. INTRODUCTION
The development of new diagnostic techniques in immunology and molecular biology during the last two decades has opened up new possibilities for rapid viral diagnosis. Solid phase immunoassays for antigen and antibody detection are now widely used in diagnostic settings. Several novel techniques have been introduced and have led to commercially available tests. Diagnostic methods using nucleic acid amplification procedures are already applied in research laboratories and will be commercialized soon. Biosensor-based diagnostic techniques have the potential of generating a result nearly instantaneously and it has become possible to monitor kinetic processes. Automatization and simplified procedures are needed to allow diagnostic tests to be performed soon after the sample has been obtained from the patient. In order to evaluate the new procedures and avoid false results, rigorous quality control in diagnostic virology will have to be instituted.
The ability to detect bacteria with the optical microscope has made rapid diagnosis in bacteriology commonplace. In virology, the need to use an electron microscope has hampered the development of diagnostic assays based on the visualization of virus particles. Besides serology, the laborious and time-consuming tissue culture approach has remained the standard method for viral diagnosis. An important step forward was the introduction of direct immunofluorescence microscopy, which is mainly used for detection of respiratory viruses [1]. Major advances in rapid diagnosis of infectious diseases were linked to the introduction of solid phase immunoassays [2] and molecular probes [3]. These techniques, although still under development, have revolutionized diagnosis of infectious diseases and brought speed and simplicity to viral diagnosis to a level similar to that found in bacteriology. Furthermore, the new techniques have also been applied in bacteriology, mycology and parasitology. The new methods allow not only the detection of pathogens, such as hepatitis and papillomaviruses that cannot be grown in culture, but also a more rapid
Correspondence to: T. Krech, In der Fina 21 a, FL-9494 Scbaan, Liechtenstein.
3f){) diagnosis. The impact of these new diagnostic methods on patient care is important, since rapid diagnosis of an infectious agent is likely to influence therapy at a time when the development of new antiviral drugs is starting to gain clinical relevance. This review will discuss the various methods based on immunological and molecular principles which are used for rapid viral diagnosis and will give an overview of the new approaches that are presently being developed. 3. IMMUNOASSAYS Immunoassays are based on specific antigenantibody interactions and have been used in the form of complement fixation and agglutination tests for about a century. The introduction of radio- and enzyme-immunoassays [2,4], which combined sensitive detection systems based on labelled reagents with solid phase adsorption, led to a considerable decrease in the time needed to complete an assay. At present, the development of these tests mainly involves a higher sensitivity by improved signal amplification systems and a greater simplicity of the tests. Examples of increasingly sensitive detection systems are the amplified enzyme reactions using enzyme-substrate cascades and cycles [5], the time-resolved fluorescence assays using europium-chelates [6] and the fluorescence- and luminescence-based reactions [7,8]. Several solid supports, like beads and magnetic particles, have been evaluated to simplify the test procedures, mainly the washing steps [6-8]. The use of microporous membranes, such as nitrocellulose or nylon, as the solid phase, was an important step towards simplification and automatization of the immunoassay-procedure [9, 10]. The recently introduced combination of this principle with coloured latex beads and membrane-chromatography made one-step immunoassays without washing procedures possible [11]. The specificity of immunoassays has been increased by replacing classical antisera by monoclonal antibodies [12] and complete antigen molecules by synthetic peptides or recombinant gene products [13].
4. N U C L E I C ACID D E T E C T I O N All techniques for nucleic acid detection rely on the spontaneous hybridization of complementary desoxyribonucleic acid strands to double stranded nucleic acid. The hybridization occurs both with DNA and RNA. With natural full length nucleic acids or, more specifically, with synthetic or cloned oligonucleotides used as molecular probes, complementary sequences of the searched nucleic acid can be detected by hybridization. The hybridization is usually detected by radioactive or enzyme markers linked to the probe. However, for viral diagnosis, these techniques lacked sensitivity, until the observation was made that nucleic acids can bc multiplied by cyclic amplification [14]. Nucleic acid is amplified in vitro by producing copies of either DNA or RNA from added nucleotides by the use of a nuclear replicase. Polymerase chain reaction (PCR) is now the most widely used technique for detection of nucleic acids. However, this technique, like all amplification procedures, is hampered by the fact that non-specific or contaminating nucleic acids are also amplified. Other methods for target amplification have been described, such as the ligase chain reaction (LCR). In the LCR, two probes hybridise along the target-DNA side by side. They are then connected with each other at the neighbouring ends by the ligase, thus forming a template DNA of the target. After separating this template DNA from the target DNA by heating (melting), it serves again as a template for probes added in excess. As the probes arc labeled, the signal is amplified simultaneously with the target DNA [15]. Another approach utilizes the amplification of the probe nucleic acid instead of the target nucleic acid. This principle is applied in the Q-beta replicase assay, where the RNA-probe is amplified by the use of a RNA-polymerase. Whereas most amplification systems are based on thermocycling, comprising heating (separation of double strands) and cooling (annealing of the primer and its elongation), the Q-beta replicase method is an isothermal single step assay producing an amplified signal within 10 to 15 min [16].
301 Another isothermal amplification procedure uses a multienzyme reaction modeled after retroviral replication. Reverse transcriptase, RNase H and a DNA-dependent polymerase are combined in one reaction. Both R N A and cDNA copies of the original target are accumulated as amplification products {17]. When molecular techniques are combined with standard procedures like tissue culture, valuable information can be obtained on disease mechanisms and on the aetiological agent. Although hybridisation assays are now available for the detection of a wide range of pathogens, several problems have limited their wide-scale application in diagnostic settings: Attempts to overcome the shortcomings include the substitution of radiolabels by non-rad.ioactive detection systems. Specificity can be increased by introducing anticontamination primers which prevent amplification of cross-contaminating nucleic acids, or by using nested primers to avoid the amplification of nucleotide sequences with low homology [18,19]. In clinical field tests, nucleic acid hybridization techniques are usually of much lower sensitivity than under ideal research laboratory conditions. Several substances have been recognized in body fluids which inhibit the amplification reaction [20]. Modified and simplified nucleic acid extraction procedures, such as those using glass powder, are therefore needed [21,22]. Solid phase based hybridization procedures, like blotting techniques [9] and sandwich-hybridization [23] or the combination with solid phase immunoassays, where nucleic acids are bound by antibodies [20] or by biotin-avidin interaction [24] on the solid phase, will probably further increase the sensitivity and specificity of detection of pathogens from body secretions and body fluids. Furthermore, the transformation of molecular procedures to the popular microtiter format will facilitate their automatization and allow large-scale investigations at affordable costs. At the moment, most commercially available molecular diagnostic procedures have not yet been sufficiently evaluated under field conditions, and they cannot be recommended as the single procedure for detecting pathogens from clinical material [25]. Ouantitation in amplified molecular assays is another
problem which limits its use for diagnostic procedures. In the case of certain diseases such as hepatitis, a full range of tests must be available to detect the various agents that can be present. The construction of broad primers that detect groups of pathogens rather than individual types, such as different enteroviruses [26], would also increase the diagnostic potential of molecular assays. When used for identification and typing of cultivated organisms, the molecular techniques have proved to be valuable tools that shorten diagnostic procedures.
5. BIOSENSORS "Biosensors are analytic microelectronic devices that use biological detector molecules (e.g., antibodies, enzymes, receptor proteins, lectins, nucleic acids) as the sensing or signal transducing element." [27]. Most biosensors use antibody-antigen-interactions (immunosensors) or immobilized enzymes. They may be used for nearly instantaneous or continuous measurement of signals derived from analyte-indicator interactions. Kinetics of antigen-antibody interactions can also be studied in vitro [28]. Although they are still at an experimental stage, biosensors open exciting prospects for rapid viral diagnosis [29,30]. Not only do immunosensor assays give an immediate result, but, in future, implantable immunosensors may allow patients at risk to be monitored on-line and in real-time for the presence of infections. As an example, transplant patients could be monitored for cytomegalovirus. Viraemia and the appearance of antibodies could be detected at an early stage and therapeutic measures could be taken before disease develops.
6. FLOW C Y T O M E T R Y Although flow cytometry gives an immediate signal, it has not been widely used to detect pathogens. There is limited experience in the detection of bacteria and only a few studies have been done with viruses [31].
302
7. P R O B L E M S Although the new techniques are rapid, sensitive and specific under controlled research laboratory conditions, viral diagnosis still is not rapid in most diagnostic settings. There are several reasons for this. Some of them have been stressed above in the discussion of nucleic acid hybridization methods. In general, there is a need for automated procedures, especially if the assay consists of several steps. On the other hand, more simplified procedures, like the single step membrane based chromatographic immunoassays, are needed. Both automated and simple tests will make it possible to run the assay at the time when the physician needs the result. As long as samples have to be collected in the laboratory to perform batched analyses once or twice a week, even the quickest assay will not give a rapid answer. Rapid diagnosis means that a result is reported within 3 to 5 h after the sample has reached the laboratory. The corresponding time for immediate diagnostic tests is 30 min. C o m p a r e d to culture, immunological and molecular direct detection methods still lack sensitivity and specificity under routine circumstances. Furthermore, field evaluations, quality control and standardisation of most direct detection assays have not yet been performed. Reference and control reagents are needed for first line evaluations and for comparison of new assays with existing methods, for quantitation of results, and for external, as well as internal, quality control of routine laboratory procedures. The sample itself is another critical point. If the collection is done improperly, it will lower the sensitivity or specificity of the assay. For molecular detection methods, samples from body sites other than the usual ones might give more significant results, as the 'supersensitive' techniques can detect a pathogen in its latent state, although this might have no clinical significance. For instance, human papilloma virus was found in certain studies in almost all cervical scrapings, when PCR was used [16]. The determination of the replicative state and the ceil-association by in situ hybridization in biopsy specimens could give more information about the clinical relevance of the result.
In conclusion, the new diagnostic techniques are potentially suited for rapid viral diagnosis on a large scale. However, a higher degree of automatization is needed. Furthermore, an extensive and permanent quality control of diagnostic procedures in virology has to be established, if the new methods are to fulfill the expectations of laboratory workers and physicians. Since the new techniques can be successfully applied in the whole field of diagnostic microbiology, this could result in a better cooperation between laboratory workers in the different subspecialities, like bacteriology, parasitology and virology.
REFERENCES [1] Gardner, P.S. and McOuillin, J, (1980) Rapid viral diagnosis: application of immunofluorescence, 2nd Edn., Butterworth, London. [2] Bidwell, D.E., Bartlett, A. and Voller, A. (1977) Enzyme immunoassays for viral diseases. J. Infect. Dis. 136, $274-$278. [3] Tenover, F.C. (1988) Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbiol. Rev. l, 82-101. [4] Feber, J.P. (1978) Radioimmunoassay in the clinical laboratory. Adv. Clin. Chem. 20, 130-161. [5] Stanley, C.J.. Johannsson, A. and Self, C.H. (1985) Enzyme amplification can enhance both the speed and the sensitivity of immunoassays. J. lmmunol. Methods 83, 89-95. [6] Walls, H . H , Johansson, K.H., Harmon, M.W., Hal,,mcn, P.E. and Kendal, A.P. (1986) Time-resolved fluoroimmunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. J. Clin. Microbiol. 24, 907-912. [7] Blackburn, G.F., Shah, H.P., Kenten, J.H., el al. (1991) Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem. 37, 1534-1539. [8] Wolf-Rogers, J., Weare, J.A, Rice, K., et al. (1990) A chemiluminescent, microparticle-membrane capture immunoassay for the detection of antibody to hepatitis B core antigen. J. Immunol. Methods 133, 191-198. [9] Proffitt, M.R. (1990) "Blotting" techniques for the diagnosis of infectious diseases. Clin. Microbiol. Newsl. 12, 121-124. [10] Abbott, G.G., Safford, J.W., MacDonald, R , Craine, M,C. and Applegren, R.R. (1990) Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection. J. Virol. Methods 27, 227-240. [11] Stratton, N.J., Hirsch, L., Harris, F., de la Maza, LM. and Peterson, E.M. (1991) Evaluation of the rapid
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[14] [15]
[16]
[17]
[18] [19]
[20]
[21]
CLEARVIEW cblamydia test for direct detection of chlamydiae from cervical specimens. J. Clin. Microbiol. 29, 1551-1553. Yolken, R.H. (1983) Use of monoclonal antibodies for viral diagnosis. Curr. Top. Micobiol. Immunol. 104, 177195. Modrow, S. and Wolf, U. (1990) Use of synthetic peptides as diagnostic reagents in virology. In: Immunochemistry of viruses. The basis for serodiagnosis and vaccines, Vol. 2 (Van Regenmortel, M.H.V. and Neurath, A.R., Eds.), pp. 81-101. Elsevier, Amsterdam Ehrlich, H.A., Gelfand, D.H. and Saiki, R.K. (1988) Specific DNA amplification. Nature 331,461-432. Hampl, H., Marshall, R.A., Perko, T. and Solomon, N. (1991) Alternative Methods for DNA probing in diagnosis: Ligase chain reaction. In: PCR Topics, (Rolfs, A., et al., Ed.), pp. 15-22. Springer Verlag, Heidelberg, Berlin. Klinger, J.D. and Pritchard, C.G. (1990) Amplified probe-based assays - possibilities and challenges in clinical microbiology. Clin. Microbiol. Newsl. 12, 133-135. Guatelli, J.C., Whi~tfield, K.M, Kwoh, D.Y., Barringer, K.J., Richman, D.D. and Gingeras, T.R. (1990) Isothermal in-vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 5, 1874-1878. Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237-238. Beyer-Finkler, E., Pfister, H. and Girardi, F. (1990) Anti-contamination primers to improve specificity of polymerase chain reaction in human papillomavirus screening (letter). Lancet 335, 1289-1290. Yolken, R.H. (1991) New methods for the quantitation of microbial nucleic acids. Pure & Appl. Chem. 63, 11271130. Loparev, V.N., Cartas, M.A., Monken, C.E., Velpandi, A. and Srinivasan, A. (1991) An efficient and simple method of DNA extraction from whole blood and cell
[22]
[23]
[24]
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[30]
[31]
lines to identify infectious agents. J. Virol. Methods 34, 105-112. Yamada, O., Matsumoto, T., Nakashima, M., et al. (1990) A new method for extracting DNA or RNA for polymerase chain reaction. J. Virol. Methods 27, 203-209. Nicholls, P.J. and Malcolm, A.D. (1989) Nucleic acid analysis by sandwich hybridisation. J. Clin. Lab. Anal. 3/2, 122-135. Harju, L., Janne, P., Kallio, A., et al. (1990) Affinity-based collection of amplified viral DNA: application to the detection of human immunodeficiency virus type 1, human cytomegalovirus and human papillomavirus type 16. Mol. Cell Probes 4, 223-235. Ehrlich, G.D. (1991) Caveats of PCR. Clin. Microbiol. News[. 13, 149-151. Zoll, G.J., Melchers, W.J.G., Kopecka, H., Jambroes, G., van der Poel, H.J.A. and Galama, J.M.D. (1992) General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections. J. Clin. Microbiol. 30, 160-165. Hunter, K.W. Jr. (1989) Biosensors. A new analytic technology for real-time, on-line biochemical monitoring. Am. J. Clin. Pathol. 91, 32-33. Karlsson, R., Altschuh, D. and Van Regenmortel, M.H.V. (1992) Measurement of antibody affinity. In: Structure of antigens (Van Regenmortel, M.H.V., Ed.), pp. 127-148. CRC Press, Boca Raton. 127-148. Parry, R.P., Love, C. and Robinson, G.A. (1990) Detection of rubella antibody using an optical immunosensor. J. Virol. Methods 27, 39-48. Dubs, M.C., Altschuh, D. and Van Regenmortel, M.H.V. (1991) Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance. Immunol. Lett. 31, 59-64. Beckmann, E. and Conolly, P. (1990) Flow cytometry: introduction and microbiological applications. Clin. Microbiol. Newsl. 12, 105-109.
299
FEMSIM 00221
Review
New techniques in rapid viral diagnosis Thomas Krech In der Fina 21 a, Schaan, Liechtenstein
Received 15 March 1992 Revision received and accepted 21 April 1992
Key words: Rapid viral diagnosis; Biosensor; Nucleic acid amplification; Polymerase chain reaction; lmmunoassay
1. SUMMARY
2. INTRODUCTION
The development of new diagnostic techniques in immunology and molecular biology during the last two decades has opened up new possibilities for rapid viral diagnosis. Solid phase immunoassays for antigen and antibody detection are now widely used in diagnostic settings. Several novel techniques have been introduced and have led to commercially available tests. Diagnostic methods using nucleic acid amplification procedures are already applied in research laboratories and will be commercialized soon. Biosensor-based diagnostic techniques have the potential of generating a result nearly instantaneously and it has become possible to monitor kinetic processes. Automatization and simplified procedures are needed to allow diagnostic tests to be performed soon after the sample has been obtained from the patient. In order to evaluate the new procedures and avoid false results, rigorous quality control in diagnostic virology will have to be instituted.
The ability to detect bacteria with the optical microscope has made rapid diagnosis in bacteriology commonplace. In virology, the need to use an electron microscope has hampered the development of diagnostic assays based on the visualization of virus particles. Besides serology, the laborious and time-consuming tissue culture approach has remained the standard method for viral diagnosis. An important step forward was the introduction of direct immunofluorescence microscopy, which is mainly used for detection of respiratory viruses [1]. Major advances in rapid diagnosis of infectious diseases were linked to the introduction of solid phase immunoassays [2] and molecular probes [3]. These techniques, although still under development, have revolutionized diagnosis of infectious diseases and brought speed and simplicity to viral diagnosis to a level similar to that found in bacteriology. Furthermore, the new techniques have also been applied in bacteriology, mycology and parasitology. The new methods allow not only the detection of pathogens, such as hepatitis and papillomaviruses that cannot be grown in culture, but also a more rapid
Correspondence to: T. Krech, In der Fina 21 a, FL-9494 Scbaan, Liechtenstein.
3f){) diagnosis. The impact of these new diagnostic methods on patient care is important, since rapid diagnosis of an infectious agent is likely to influence therapy at a time when the development of new antiviral drugs is starting to gain clinical relevance. This review will discuss the various methods based on immunological and molecular principles which are used for rapid viral diagnosis and will give an overview of the new approaches that are presently being developed. 3. IMMUNOASSAYS Immunoassays are based on specific antigenantibody interactions and have been used in the form of complement fixation and agglutination tests for about a century. The introduction of radio- and enzyme-immunoassays [2,4], which combined sensitive detection systems based on labelled reagents with solid phase adsorption, led to a considerable decrease in the time needed to complete an assay. At present, the development of these tests mainly involves a higher sensitivity by improved signal amplification systems and a greater simplicity of the tests. Examples of increasingly sensitive detection systems are the amplified enzyme reactions using enzyme-substrate cascades and cycles [5], the time-resolved fluorescence assays using europium-chelates [6] and the fluorescence- and luminescence-based reactions [7,8]. Several solid supports, like beads and magnetic particles, have been evaluated to simplify the test procedures, mainly the washing steps [6-8]. The use of microporous membranes, such as nitrocellulose or nylon, as the solid phase, was an important step towards simplification and automatization of the immunoassay-procedure [9, 10]. The recently introduced combination of this principle with coloured latex beads and membrane-chromatography made one-step immunoassays without washing procedures possible [11]. The specificity of immunoassays has been increased by replacing classical antisera by monoclonal antibodies [12] and complete antigen molecules by synthetic peptides or recombinant gene products [13].
4. N U C L E I C ACID D E T E C T I O N All techniques for nucleic acid detection rely on the spontaneous hybridization of complementary desoxyribonucleic acid strands to double stranded nucleic acid. The hybridization occurs both with DNA and RNA. With natural full length nucleic acids or, more specifically, with synthetic or cloned oligonucleotides used as molecular probes, complementary sequences of the searched nucleic acid can be detected by hybridization. The hybridization is usually detected by radioactive or enzyme markers linked to the probe. However, for viral diagnosis, these techniques lacked sensitivity, until the observation was made that nucleic acids can bc multiplied by cyclic amplification [14]. Nucleic acid is amplified in vitro by producing copies of either DNA or RNA from added nucleotides by the use of a nuclear replicase. Polymerase chain reaction (PCR) is now the most widely used technique for detection of nucleic acids. However, this technique, like all amplification procedures, is hampered by the fact that non-specific or contaminating nucleic acids are also amplified. Other methods for target amplification have been described, such as the ligase chain reaction (LCR). In the LCR, two probes hybridise along the target-DNA side by side. They are then connected with each other at the neighbouring ends by the ligase, thus forming a template DNA of the target. After separating this template DNA from the target DNA by heating (melting), it serves again as a template for probes added in excess. As the probes arc labeled, the signal is amplified simultaneously with the target DNA [15]. Another approach utilizes the amplification of the probe nucleic acid instead of the target nucleic acid. This principle is applied in the Q-beta replicase assay, where the RNA-probe is amplified by the use of a RNA-polymerase. Whereas most amplification systems are based on thermocycling, comprising heating (separation of double strands) and cooling (annealing of the primer and its elongation), the Q-beta replicase method is an isothermal single step assay producing an amplified signal within 10 to 15 min [16].
301 Another isothermal amplification procedure uses a multienzyme reaction modeled after retroviral replication. Reverse transcriptase, RNase H and a DNA-dependent polymerase are combined in one reaction. Both R N A and cDNA copies of the original target are accumulated as amplification products {17]. When molecular techniques are combined with standard procedures like tissue culture, valuable information can be obtained on disease mechanisms and on the aetiological agent. Although hybridisation assays are now available for the detection of a wide range of pathogens, several problems have limited their wide-scale application in diagnostic settings: Attempts to overcome the shortcomings include the substitution of radiolabels by non-rad.ioactive detection systems. Specificity can be increased by introducing anticontamination primers which prevent amplification of cross-contaminating nucleic acids, or by using nested primers to avoid the amplification of nucleotide sequences with low homology [18,19]. In clinical field tests, nucleic acid hybridization techniques are usually of much lower sensitivity than under ideal research laboratory conditions. Several substances have been recognized in body fluids which inhibit the amplification reaction [20]. Modified and simplified nucleic acid extraction procedures, such as those using glass powder, are therefore needed [21,22]. Solid phase based hybridization procedures, like blotting techniques [9] and sandwich-hybridization [23] or the combination with solid phase immunoassays, where nucleic acids are bound by antibodies [20] or by biotin-avidin interaction [24] on the solid phase, will probably further increase the sensitivity and specificity of detection of pathogens from body secretions and body fluids. Furthermore, the transformation of molecular procedures to the popular microtiter format will facilitate their automatization and allow large-scale investigations at affordable costs. At the moment, most commercially available molecular diagnostic procedures have not yet been sufficiently evaluated under field conditions, and they cannot be recommended as the single procedure for detecting pathogens from clinical material [25]. Ouantitation in amplified molecular assays is another
problem which limits its use for diagnostic procedures. In the case of certain diseases such as hepatitis, a full range of tests must be available to detect the various agents that can be present. The construction of broad primers that detect groups of pathogens rather than individual types, such as different enteroviruses [26], would also increase the diagnostic potential of molecular assays. When used for identification and typing of cultivated organisms, the molecular techniques have proved to be valuable tools that shorten diagnostic procedures.
5. BIOSENSORS "Biosensors are analytic microelectronic devices that use biological detector molecules (e.g., antibodies, enzymes, receptor proteins, lectins, nucleic acids) as the sensing or signal transducing element." [27]. Most biosensors use antibody-antigen-interactions (immunosensors) or immobilized enzymes. They may be used for nearly instantaneous or continuous measurement of signals derived from analyte-indicator interactions. Kinetics of antigen-antibody interactions can also be studied in vitro [28]. Although they are still at an experimental stage, biosensors open exciting prospects for rapid viral diagnosis [29,30]. Not only do immunosensor assays give an immediate result, but, in future, implantable immunosensors may allow patients at risk to be monitored on-line and in real-time for the presence of infections. As an example, transplant patients could be monitored for cytomegalovirus. Viraemia and the appearance of antibodies could be detected at an early stage and therapeutic measures could be taken before disease develops.
6. FLOW C Y T O M E T R Y Although flow cytometry gives an immediate signal, it has not been widely used to detect pathogens. There is limited experience in the detection of bacteria and only a few studies have been done with viruses [31].
302
7. P R O B L E M S Although the new techniques are rapid, sensitive and specific under controlled research laboratory conditions, viral diagnosis still is not rapid in most diagnostic settings. There are several reasons for this. Some of them have been stressed above in the discussion of nucleic acid hybridization methods. In general, there is a need for automated procedures, especially if the assay consists of several steps. On the other hand, more simplified procedures, like the single step membrane based chromatographic immunoassays, are needed. Both automated and simple tests will make it possible to run the assay at the time when the physician needs the result. As long as samples have to be collected in the laboratory to perform batched analyses once or twice a week, even the quickest assay will not give a rapid answer. Rapid diagnosis means that a result is reported within 3 to 5 h after the sample has reached the laboratory. The corresponding time for immediate diagnostic tests is 30 min. C o m p a r e d to culture, immunological and molecular direct detection methods still lack sensitivity and specificity under routine circumstances. Furthermore, field evaluations, quality control and standardisation of most direct detection assays have not yet been performed. Reference and control reagents are needed for first line evaluations and for comparison of new assays with existing methods, for quantitation of results, and for external, as well as internal, quality control of routine laboratory procedures. The sample itself is another critical point. If the collection is done improperly, it will lower the sensitivity or specificity of the assay. For molecular detection methods, samples from body sites other than the usual ones might give more significant results, as the 'supersensitive' techniques can detect a pathogen in its latent state, although this might have no clinical significance. For instance, human papilloma virus was found in certain studies in almost all cervical scrapings, when PCR was used [16]. The determination of the replicative state and the ceil-association by in situ hybridization in biopsy specimens could give more information about the clinical relevance of the result.
In conclusion, the new diagnostic techniques are potentially suited for rapid viral diagnosis on a large scale. However, a higher degree of automatization is needed. Furthermore, an extensive and permanent quality control of diagnostic procedures in virology has to be established, if the new methods are to fulfill the expectations of laboratory workers and physicians. Since the new techniques can be successfully applied in the whole field of diagnostic microbiology, this could result in a better cooperation between laboratory workers in the different subspecialities, like bacteriology, parasitology and virology.
REFERENCES [1] Gardner, P.S. and McOuillin, J, (1980) Rapid viral diagnosis: application of immunofluorescence, 2nd Edn., Butterworth, London. [2] Bidwell, D.E., Bartlett, A. and Voller, A. (1977) Enzyme immunoassays for viral diseases. J. Infect. Dis. 136, $274-$278. [3] Tenover, F.C. (1988) Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbiol. Rev. l, 82-101. [4] Feber, J.P. (1978) Radioimmunoassay in the clinical laboratory. Adv. Clin. Chem. 20, 130-161. [5] Stanley, C.J.. Johannsson, A. and Self, C.H. (1985) Enzyme amplification can enhance both the speed and the sensitivity of immunoassays. J. lmmunol. Methods 83, 89-95. [6] Walls, H . H , Johansson, K.H., Harmon, M.W., Hal,,mcn, P.E. and Kendal, A.P. (1986) Time-resolved fluoroimmunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. J. Clin. Microbiol. 24, 907-912. [7] Blackburn, G.F., Shah, H.P., Kenten, J.H., el al. (1991) Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem. 37, 1534-1539. [8] Wolf-Rogers, J., Weare, J.A, Rice, K., et al. (1990) A chemiluminescent, microparticle-membrane capture immunoassay for the detection of antibody to hepatitis B core antigen. J. Immunol. Methods 133, 191-198. [9] Proffitt, M.R. (1990) "Blotting" techniques for the diagnosis of infectious diseases. Clin. Microbiol. Newsl. 12, 121-124. [10] Abbott, G.G., Safford, J.W., MacDonald, R , Craine, M,C. and Applegren, R.R. (1990) Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection. J. Virol. Methods 27, 227-240. [11] Stratton, N.J., Hirsch, L., Harris, F., de la Maza, LM. and Peterson, E.M. (1991) Evaluation of the rapid
303
[12]
[13]
[14] [15]
[16]
[17]
[18] [19]
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