- Email: [email protected]
Molecular methods for diagnosis of influenza
International Congress Series 1219 (2001) 267 – 273
Molecular methods for diagnosis of influenza Maria C. Zambon*, Joanna S. Ellis Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, Public Health Laboratory Service, 61 Colindale Avenue, Colindale, London NW9 5HT, UK
Abstract The accurate and reliable diagnosis of transmissible diseases is the first requirement to ensure their control. Several different pathogens can produce respiratory illness with similar clinical symptoms, making an accurate diagnosis of influenza by a physician difficult. The invention and development of polymerase chain reaction (PCR) technology has enabled rapid and sensitive viral diagnostic tests to influence patient treatment. Molecular methods used directly on clinical materials have an important role to play in the diagnosis and surveillance of influenza viruses. D 2001 Elsevier Science B.V. All rights reserved. Keywords: RT-PCR; Sequence; HMA; Surveillance; Restriction assay
1. Introduction The application of molecular methodology has had an important impact on the diagnosis and surveillance of influenza viruses. Since influenza viruses continue to circulate and cause significant morbidity and mortality throughout the world, accurate identification and monitoring of circulating strains are essential. The early detection and characterisation of newly emerging variants is one of the aims of the WHO global surveillance network. Both timely and accurate information on the relationship of circulating viruses to current vaccine strains aid optimal vaccine formulation. The sensitivity and specificity of molecular methods such as reverse-transcription polymerase chain reaction (RT-PCR) assays enable the detailed analysis of the molecular epidemiology of circulating strains. While determination of changes in the sequence of the
*
Corresponding author. Tel.: +44-208-200-4400. E-mail address: [email protected] (M.C. Zambon).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 3 8 - 7
268
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
haemagglutinin (HA) gene can give us information on the direction of genetic drift, the effect of specific amino acid changes on antigenicity cannot as yet be predicted. Therefore, virus isolation followed by full characterisation of the antigenic properties of virus isolates still remains the cornerstone of WHO influenza virus surveillance.
2. Application of molecular methods 2.1. Diagnosis The advent of molecular technology has transformed the diagnosis of a number of diseases, for example HIV and HCV infections [1,2]. This is in contrast to the diagnosis of respiratory infections, where the application of molecular methods to the detection of respiratory pathogens is still in its infancy. The development of the new neuraminidase inhibitors and the subsequent evaluation of these drugs in clinical trials have provided an excellent source of data on the usefulness of PCR in the diagnosis of influenza infection [3]. In some early phase III trials of zanamivir in Europe and North America, samples were collected from community cases of influenza during periods when influenza virus was circulating from patients who presented within the first or second calendar day of onset of symptoms. Patients were aged between 12 and 81 years (mean 37 years) and had fever together with two symptoms (headache, myalgia, sore throat, cough). The results of the comparison of the percentage of samples positive for influenza A or B virus by virus isolation, serology or PCR show an excellent concordance between PCR and the classical methodology (Fig. 1). Furthermore, there was a significant correlation between the number of tests positive and illness severity. Where all three tests were positive, there was a significant correlation between duration of illness, but not antibiotic use and the risk of complications (data not shown). Although PCR was more sensitive than either culture or paired haemagglutina-
Fig. 1. Concordance of diagnostic test results in 791 patients with positive results from serology, virus culture or PCR.
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
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tion inhibition (HI) serology, PCR positivity alone was not more likely to be associated with severity of illness, development of complications or antibiotic usage. From these analyses, it can be concluded that PCR provides rapid and accurate diagnosis in individual patient diagnosis and is the most sensitive method for detection of cases of influenza in the community. 2.2. Surveillance The detection and analysis of influenza viruses by molecular methods also have an important role to play in enhancing the surveillance and characterisation of circulating influenza strains. In some national surveillance schemes, the benefit of utilising multiplex RT-PCR has been demonstrated successfully [4]. In Portugal, a comparison of multiplex RT-PCR for the detection of influenza viruses with culture, enzyme immunoassay (EIA) and serology was performed during surveillance over seven influenza seasons from 1992 to 1999 [5]. More samples were found to be positive by RT-PCR than by any of the other methods used. The best correlation was seen between RT-PCR and paired serology, where 43.6% and 38%, respectively, of the total number of individuals sampled were positive for influenza virus, which also reflected the clinical indices of influenza-like illness in the community. The annual outbreak of influenza activity in Scotland is monitored by a communitybased surveillance scheme and from 2000 – 2001, virological surveillance will be performed by molecular detection alone. This decision was made following the results of a comparison of RT-PCR with culture and serology, which was recently reported [6]. An excellent concordance between diagnosis by RT-PCR and serology was demonstrated, with influenza virus detected in 57% and 61% of samples, respectively. However, whereas, RT-PCR is rapid and can be performed in 36 h, serological methods are usually retrospective. It can be concluded that the use of RT-PCR in surveillance of influenza is more sensitive and rapid than established methods. Molecular methods are able to give a better estimate of the true burden of illness due to influenza in the community. In addition, since multiplex approaches to molecular diagnosis are able to detect more than one respiratory pathogen in a single specimen [7,8], they are more cost-effective than singletarget assays. The exact impact of molecular methodology on surveillance is likely to depend upon the sensitivity of laboratory systems already in place. In both of the above national schemes, culture of influenza viruses was found to be difficult and suboptimal for a number of reasons. Hence, the decision as to whether to use molecular methods, either in addition to or in the place of traditional assays has to be made by each laboratory in each country according to local circumstances.
3. Outbreaks The investigations of outbreaks of respiratory illness are often hampered by the inability to culture the organism responsible. However, the use of molecular techniques directly on clinical respiratory specimens facilitates the analysis of respiratory outbreaks
270
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
Fig. 2. Comparison of detection of influenza virus by RT-PCR and cell culture in specimens from an outbreak of respiratory illness on a cruise-ship. Lanes 1 – 7 and 9 – 16; clinical specimens. Lanes 8, 17, 21; negative controls. Lane 18; A H1N1 positive control. Lane 19; A H3N2 positive control. Lane 20; influenza B positive control. Mw; DNA molecular weight markers.
and can provide rapid identification of an organism and reveal its relationship to currently circulating viruses and vaccine strains. This was demonstrated most recently during the investigation of an outbreak of influenza-like illness on board a cruise-ship in the Mediterranean in 1999 [9]. A total of 55 of 490 crew members and 60 of 590 passengers presented with respiratory tract infection. Of the passengers, two were hospitalised with pneumonia. Respiratory samples were analysed by RT-PCR and culture within 24 h of collection. Of the first 15 throat swabs tested, 13 were positive by RT-PCR for influenza AH3N2 virus (Fig. 2), whereas, virus was isolated by culture from only four of the RTPCR positive samples. On the basis of these findings, the decision was made to immunise the crew with influenza vaccine. These results highlight the suitability of applying molecular methodology to outbreak situations.
4. Analysis of molecular products The coupling of amplification by PCR of nucleic acid directly from respiratory samples with typing techniques allows the analysis of circulating lineages of virus genes and enhances rapid tracking of influenza virus evolution. A combination of RTPCR and enzyme digestion of the PCR product (PCR restriction assay) has been used to differentiate rapidly genetic variants that are antigenically similar [10]. This technique may also differentiate between vaccine strains and currently circulating strains. PCR restriction assays have most recently been used to differentiate the internal genes of human influenza H1N1, H3N2 and H5N1 influenza A viruses [11].
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
271
Fig. 3. Characterisation of influenza M gene PCR products by heteroduplex mobility assay (HMA). Lane 1; molecular weight markers, Lane 2; reference PCR amplicon alone (homoduplex), Lanes 3 – 9; reference PCR amplicon mixed, denatured and annealed with test amplicons of decreasing divergence, which form heteroduplexes of increasing mobility with the reference DNA.
Direct amplification of internal genes from clinical material can also be coupled with heteroduplex mobility assays (HMA) to allow rapid species identification of the origin of influenza viruses, or analysis of variant strains (Fig. 3). Sequence analysis of PCR amplicons is routinely performed in many laboratories, particularly on HA gene products where an association between sequence changes with genetic drift is studied [12,13].
5. Tissue diagnosis The most important application of RT-PCR and sequencing in tissue analysis to date has been in the genetic analysis of the 1918 pandemic influenza strain in archival material [14]. Influenza RNA fragments were isolated from lung tissue from three victims of the 1918 pandemic and the coding sequences of the genes are being analysed to determine the origin of this virus. Molecular methodology is of particular use in the study of postmortem specimens (Fig. 4) and may be of extreme value in the diagnosis of influenza in the central nervous system [15,16]. Analysis of other body tissues may help to clarify mechanisms of pathogenesis [17].
6. Approaches When designing a molecular diagnostic assay, the prospective application of the assay will influence the choice of gene target. For type-specific diagnosis of influenza A, B or C infection, internal genes such as nucleoprotein (NP) and matrix (M) genes
272
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
Fig. 4. Detection of influenza AH3N2 by RT-PCR on post-mortem lung tissue samples. Lanes 1 and 2; tissue samples. Lane 3; negative control. Lanes 4 – 6; influenza A H1N1, A H3N2 and B positive controls, respectively.
are usually chosen, since these are highly conserved within influenza types [18]. If, however, information on the subtype of influenza A is required, then the genes encoding the surface antigens must be targeted. One advantage of assays based on detection of the HA gene is that subsequent sequence information can be determined from the assay products. Multisegment PCR using primers complimentary to the conserved 50 and 30 sequences on each gene allows the detection of all segments in a single reaction [19].
7. Predictions for the future Diagnostic molecular methodology has developed considerably during the past few years. The coupling of automated purification of nucleic acids together with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. Quantitative RT-PCR systems are being designed to allow the determination of viral load in infections. Finally, the use of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to revolutionise influenza diagnosis. Although molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they will continue to be invaluable in aiding our understanding of the epidemiology of influenza viruses.
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
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Acknowledgements We would like to acknowledge the excellent work of Carol Sadler and Alex Elliot.
References [1] K. Christano, A.M. DiBisceglie, J.H. Hoofnagle, S.M. Feinstone, Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets, Hepatology 14 (1991) 51 – 55. [2] S. Cassol, A. Butcher, S. Kinard, J. Spadoro, N. Lapointe, S. Read, et al., Rapid screening for the early detection of mother-to-child transmission of HIV-1, Journal of Clinical Microbiology 32 (1994) 2641 – 2645. [3] M.C. Zambon, J. Hays, A. Webster, R. Newman, Comparison of virus culture, RT-PCR and serology in the diagnosis of influenza, Journal of Antimicrobial Chemotherapy 44 (Supplement A) (1999) 42. [4] J.S. Ellis, D.M. Fleming, M.C. Zambon, Multiplex reverse transcription-PCR for surveillance of influenza A and B viruses in England and Wales in 1995 and 1996, Journal of Clinical Microbiology 35 (8) (1997) 2076 – 2082. [5] H. Rebelo-de-Andrade, M.C. Zambon, Different diagnostic methods for detection of influenza epidemics, Epidemiology and Infection 124 (3) (2000) 515 – 522. [6] W.F. Carman, J. Walker, S. McInyyre, A. Noone, P. Christie, J. Millar, et al., Rapid virological surveillance of community influenza infection in general practice, British Medical Journal 321 (2000) 736 – 737. [7] J. Stockton, J.S. Ellis, M. Saville, J.P. Clewley, M.C. Zambon, Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses, Journal of Clinical Microbiology 36 (1998) 2990 – 2995. [8] B. Grondahl, W. Puppe, A. Hoppe, I. Kuhne, J.A. Weighl, H.J. Schmitt, Rapid identification of nine microorganisms causing respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study, Journal of Clinical Microbiology 37 (1999) 1 – 7. [9] Anonymous. Influenza on a cruise ship in the Mediterranean, Communicable Disease Report 9 (24) (1999) 209, 12. [10] J.S. Ellis, C.J. Sadler, P. Laidler, H. Rebelo-de-Andrade, M.C. Zambon, Analysis of influenza A H3N2 strains isolated in England during 1995 – 1996 using polymerase chain reaction restriction, Journal of Medical Virology 51 (1997) 234 – 241. [11] L.A. Cooper, K. Subbarao, A simple restriction fragment length polymorphism-based strategy that can distinguish the internal genes of human H1N1, H3N2, and H5N1 influenza A viruses, Journal of Clinical Microbiology 38 (7) (2000) 2579 – 2583. [12] N. Cox, C. Bender, Molecular epidemiology of influenza, Seminars in Virology 6 (1995) 359 – 370. [13] J.S. Ellis, P. Chakraverty, J.P. Clewley, Genetic and antigenic variation in the haemagglutinin of recently circulating influenza A (H3N2) viruses in the United Kingdom, Archives of Virology 140 (1995) 1889 – 1904. [14] J.K. Taubenberger, A.H. Reid, A.E. Krafft, K.E. Bijwaard, T.G. Fanning, Initial genetic characterisation of the 1918 ‘‘Spanish’’ influenza virus, Science 275 (5307) (1997) 1793 – 1796. [15] S. Fujimoto, M. Kobayashi, O. Uemura, M. Iwasa, T. Ando, T. Katoh, et al., PCR on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis, The Lancet 352 (1998) 873 – 875. [16] J.A. McCullers, S. Facchini, P.J. Chesney, R.G. Webster, Influenza B virus encephalitis, Clinical Infectious Diseases 28 (4) (1999) 898 – 900. [17] H. Xu, O. Yasui, H. Tsuruoka, K. Kuroda, K. Hayashi, A. Yamada, et al., Isolation of type B influenza virus from the blood of children, Clinical Infectious Diseases 27 (1998) 654 – 655. [18] E.J.C. Claas, M.J.W. Sprenger, G.E.M. Kleter, R. van Beek, W.G.V. Quint, N. Masurel, Type-specific identification of influenza viruses A, B and C by the polymerase chain reaction, Journal of Virological Methods 39 (1992) 1 – 13. [19] D.E. Wentworth, M. McGregor, M.D. Macklin, V. Neumann, V.S. Hinshaw, Transmission of swine influenza virus to humans after exposure to experimentally infected pigs, Journal of Infectious Diseases 175 (1997) 7 – 15.
Molecular methods for diagnosis of influenza Maria C. Zambon*, Joanna S. Ellis Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, Public Health Laboratory Service, 61 Colindale Avenue, Colindale, London NW9 5HT, UK
Abstract The accurate and reliable diagnosis of transmissible diseases is the first requirement to ensure their control. Several different pathogens can produce respiratory illness with similar clinical symptoms, making an accurate diagnosis of influenza by a physician difficult. The invention and development of polymerase chain reaction (PCR) technology has enabled rapid and sensitive viral diagnostic tests to influence patient treatment. Molecular methods used directly on clinical materials have an important role to play in the diagnosis and surveillance of influenza viruses. D 2001 Elsevier Science B.V. All rights reserved. Keywords: RT-PCR; Sequence; HMA; Surveillance; Restriction assay
1. Introduction The application of molecular methodology has had an important impact on the diagnosis and surveillance of influenza viruses. Since influenza viruses continue to circulate and cause significant morbidity and mortality throughout the world, accurate identification and monitoring of circulating strains are essential. The early detection and characterisation of newly emerging variants is one of the aims of the WHO global surveillance network. Both timely and accurate information on the relationship of circulating viruses to current vaccine strains aid optimal vaccine formulation. The sensitivity and specificity of molecular methods such as reverse-transcription polymerase chain reaction (RT-PCR) assays enable the detailed analysis of the molecular epidemiology of circulating strains. While determination of changes in the sequence of the
*
Corresponding author. Tel.: +44-208-200-4400. E-mail address: [email protected] (M.C. Zambon).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 3 8 - 7
268
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
haemagglutinin (HA) gene can give us information on the direction of genetic drift, the effect of specific amino acid changes on antigenicity cannot as yet be predicted. Therefore, virus isolation followed by full characterisation of the antigenic properties of virus isolates still remains the cornerstone of WHO influenza virus surveillance.
2. Application of molecular methods 2.1. Diagnosis The advent of molecular technology has transformed the diagnosis of a number of diseases, for example HIV and HCV infections [1,2]. This is in contrast to the diagnosis of respiratory infections, where the application of molecular methods to the detection of respiratory pathogens is still in its infancy. The development of the new neuraminidase inhibitors and the subsequent evaluation of these drugs in clinical trials have provided an excellent source of data on the usefulness of PCR in the diagnosis of influenza infection [3]. In some early phase III trials of zanamivir in Europe and North America, samples were collected from community cases of influenza during periods when influenza virus was circulating from patients who presented within the first or second calendar day of onset of symptoms. Patients were aged between 12 and 81 years (mean 37 years) and had fever together with two symptoms (headache, myalgia, sore throat, cough). The results of the comparison of the percentage of samples positive for influenza A or B virus by virus isolation, serology or PCR show an excellent concordance between PCR and the classical methodology (Fig. 1). Furthermore, there was a significant correlation between the number of tests positive and illness severity. Where all three tests were positive, there was a significant correlation between duration of illness, but not antibiotic use and the risk of complications (data not shown). Although PCR was more sensitive than either culture or paired haemagglutina-
Fig. 1. Concordance of diagnostic test results in 791 patients with positive results from serology, virus culture or PCR.
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
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tion inhibition (HI) serology, PCR positivity alone was not more likely to be associated with severity of illness, development of complications or antibiotic usage. From these analyses, it can be concluded that PCR provides rapid and accurate diagnosis in individual patient diagnosis and is the most sensitive method for detection of cases of influenza in the community. 2.2. Surveillance The detection and analysis of influenza viruses by molecular methods also have an important role to play in enhancing the surveillance and characterisation of circulating influenza strains. In some national surveillance schemes, the benefit of utilising multiplex RT-PCR has been demonstrated successfully [4]. In Portugal, a comparison of multiplex RT-PCR for the detection of influenza viruses with culture, enzyme immunoassay (EIA) and serology was performed during surveillance over seven influenza seasons from 1992 to 1999 [5]. More samples were found to be positive by RT-PCR than by any of the other methods used. The best correlation was seen between RT-PCR and paired serology, where 43.6% and 38%, respectively, of the total number of individuals sampled were positive for influenza virus, which also reflected the clinical indices of influenza-like illness in the community. The annual outbreak of influenza activity in Scotland is monitored by a communitybased surveillance scheme and from 2000 – 2001, virological surveillance will be performed by molecular detection alone. This decision was made following the results of a comparison of RT-PCR with culture and serology, which was recently reported [6]. An excellent concordance between diagnosis by RT-PCR and serology was demonstrated, with influenza virus detected in 57% and 61% of samples, respectively. However, whereas, RT-PCR is rapid and can be performed in 36 h, serological methods are usually retrospective. It can be concluded that the use of RT-PCR in surveillance of influenza is more sensitive and rapid than established methods. Molecular methods are able to give a better estimate of the true burden of illness due to influenza in the community. In addition, since multiplex approaches to molecular diagnosis are able to detect more than one respiratory pathogen in a single specimen [7,8], they are more cost-effective than singletarget assays. The exact impact of molecular methodology on surveillance is likely to depend upon the sensitivity of laboratory systems already in place. In both of the above national schemes, culture of influenza viruses was found to be difficult and suboptimal for a number of reasons. Hence, the decision as to whether to use molecular methods, either in addition to or in the place of traditional assays has to be made by each laboratory in each country according to local circumstances.
3. Outbreaks The investigations of outbreaks of respiratory illness are often hampered by the inability to culture the organism responsible. However, the use of molecular techniques directly on clinical respiratory specimens facilitates the analysis of respiratory outbreaks
270
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
Fig. 2. Comparison of detection of influenza virus by RT-PCR and cell culture in specimens from an outbreak of respiratory illness on a cruise-ship. Lanes 1 – 7 and 9 – 16; clinical specimens. Lanes 8, 17, 21; negative controls. Lane 18; A H1N1 positive control. Lane 19; A H3N2 positive control. Lane 20; influenza B positive control. Mw; DNA molecular weight markers.
and can provide rapid identification of an organism and reveal its relationship to currently circulating viruses and vaccine strains. This was demonstrated most recently during the investigation of an outbreak of influenza-like illness on board a cruise-ship in the Mediterranean in 1999 [9]. A total of 55 of 490 crew members and 60 of 590 passengers presented with respiratory tract infection. Of the passengers, two were hospitalised with pneumonia. Respiratory samples were analysed by RT-PCR and culture within 24 h of collection. Of the first 15 throat swabs tested, 13 were positive by RT-PCR for influenza AH3N2 virus (Fig. 2), whereas, virus was isolated by culture from only four of the RTPCR positive samples. On the basis of these findings, the decision was made to immunise the crew with influenza vaccine. These results highlight the suitability of applying molecular methodology to outbreak situations.
4. Analysis of molecular products The coupling of amplification by PCR of nucleic acid directly from respiratory samples with typing techniques allows the analysis of circulating lineages of virus genes and enhances rapid tracking of influenza virus evolution. A combination of RTPCR and enzyme digestion of the PCR product (PCR restriction assay) has been used to differentiate rapidly genetic variants that are antigenically similar [10]. This technique may also differentiate between vaccine strains and currently circulating strains. PCR restriction assays have most recently been used to differentiate the internal genes of human influenza H1N1, H3N2 and H5N1 influenza A viruses [11].
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
271
Fig. 3. Characterisation of influenza M gene PCR products by heteroduplex mobility assay (HMA). Lane 1; molecular weight markers, Lane 2; reference PCR amplicon alone (homoduplex), Lanes 3 – 9; reference PCR amplicon mixed, denatured and annealed with test amplicons of decreasing divergence, which form heteroduplexes of increasing mobility with the reference DNA.
Direct amplification of internal genes from clinical material can also be coupled with heteroduplex mobility assays (HMA) to allow rapid species identification of the origin of influenza viruses, or analysis of variant strains (Fig. 3). Sequence analysis of PCR amplicons is routinely performed in many laboratories, particularly on HA gene products where an association between sequence changes with genetic drift is studied [12,13].
5. Tissue diagnosis The most important application of RT-PCR and sequencing in tissue analysis to date has been in the genetic analysis of the 1918 pandemic influenza strain in archival material [14]. Influenza RNA fragments were isolated from lung tissue from three victims of the 1918 pandemic and the coding sequences of the genes are being analysed to determine the origin of this virus. Molecular methodology is of particular use in the study of postmortem specimens (Fig. 4) and may be of extreme value in the diagnosis of influenza in the central nervous system [15,16]. Analysis of other body tissues may help to clarify mechanisms of pathogenesis [17].
6. Approaches When designing a molecular diagnostic assay, the prospective application of the assay will influence the choice of gene target. For type-specific diagnosis of influenza A, B or C infection, internal genes such as nucleoprotein (NP) and matrix (M) genes
272
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
Fig. 4. Detection of influenza AH3N2 by RT-PCR on post-mortem lung tissue samples. Lanes 1 and 2; tissue samples. Lane 3; negative control. Lanes 4 – 6; influenza A H1N1, A H3N2 and B positive controls, respectively.
are usually chosen, since these are highly conserved within influenza types [18]. If, however, information on the subtype of influenza A is required, then the genes encoding the surface antigens must be targeted. One advantage of assays based on detection of the HA gene is that subsequent sequence information can be determined from the assay products. Multisegment PCR using primers complimentary to the conserved 50 and 30 sequences on each gene allows the detection of all segments in a single reaction [19].
7. Predictions for the future Diagnostic molecular methodology has developed considerably during the past few years. The coupling of automated purification of nucleic acids together with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. Quantitative RT-PCR systems are being designed to allow the determination of viral load in infections. Finally, the use of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to revolutionise influenza diagnosis. Although molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they will continue to be invaluable in aiding our understanding of the epidemiology of influenza viruses.
M.C. Zambon, J.S. Ellis / International Congress Series 1219 (2001) 267–273
273
Acknowledgements We would like to acknowledge the excellent work of Carol Sadler and Alex Elliot.
References [1] K. Christano, A.M. DiBisceglie, J.H. Hoofnagle, S.M. Feinstone, Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets, Hepatology 14 (1991) 51 – 55. [2] S. Cassol, A. Butcher, S. Kinard, J. Spadoro, N. Lapointe, S. Read, et al., Rapid screening for the early detection of mother-to-child transmission of HIV-1, Journal of Clinical Microbiology 32 (1994) 2641 – 2645. [3] M.C. Zambon, J. Hays, A. Webster, R. Newman, Comparison of virus culture, RT-PCR and serology in the diagnosis of influenza, Journal of Antimicrobial Chemotherapy 44 (Supplement A) (1999) 42. [4] J.S. Ellis, D.M. Fleming, M.C. Zambon, Multiplex reverse transcription-PCR for surveillance of influenza A and B viruses in England and Wales in 1995 and 1996, Journal of Clinical Microbiology 35 (8) (1997) 2076 – 2082. [5] H. Rebelo-de-Andrade, M.C. Zambon, Different diagnostic methods for detection of influenza epidemics, Epidemiology and Infection 124 (3) (2000) 515 – 522. [6] W.F. Carman, J. Walker, S. McInyyre, A. Noone, P. Christie, J. Millar, et al., Rapid virological surveillance of community influenza infection in general practice, British Medical Journal 321 (2000) 736 – 737. [7] J. Stockton, J.S. Ellis, M. Saville, J.P. Clewley, M.C. Zambon, Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses, Journal of Clinical Microbiology 36 (1998) 2990 – 2995. [8] B. Grondahl, W. Puppe, A. Hoppe, I. Kuhne, J.A. Weighl, H.J. Schmitt, Rapid identification of nine microorganisms causing respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study, Journal of Clinical Microbiology 37 (1999) 1 – 7. [9] Anonymous. Influenza on a cruise ship in the Mediterranean, Communicable Disease Report 9 (24) (1999) 209, 12. [10] J.S. Ellis, C.J. Sadler, P. Laidler, H. Rebelo-de-Andrade, M.C. Zambon, Analysis of influenza A H3N2 strains isolated in England during 1995 – 1996 using polymerase chain reaction restriction, Journal of Medical Virology 51 (1997) 234 – 241. [11] L.A. Cooper, K. Subbarao, A simple restriction fragment length polymorphism-based strategy that can distinguish the internal genes of human H1N1, H3N2, and H5N1 influenza A viruses, Journal of Clinical Microbiology 38 (7) (2000) 2579 – 2583. [12] N. Cox, C. Bender, Molecular epidemiology of influenza, Seminars in Virology 6 (1995) 359 – 370. [13] J.S. Ellis, P. Chakraverty, J.P. Clewley, Genetic and antigenic variation in the haemagglutinin of recently circulating influenza A (H3N2) viruses in the United Kingdom, Archives of Virology 140 (1995) 1889 – 1904. [14] J.K. Taubenberger, A.H. Reid, A.E. Krafft, K.E. Bijwaard, T.G. Fanning, Initial genetic characterisation of the 1918 ‘‘Spanish’’ influenza virus, Science 275 (5307) (1997) 1793 – 1796. [15] S. Fujimoto, M. Kobayashi, O. Uemura, M. Iwasa, T. Ando, T. Katoh, et al., PCR on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis, The Lancet 352 (1998) 873 – 875. [16] J.A. McCullers, S. Facchini, P.J. Chesney, R.G. Webster, Influenza B virus encephalitis, Clinical Infectious Diseases 28 (4) (1999) 898 – 900. [17] H. Xu, O. Yasui, H. Tsuruoka, K. Kuroda, K. Hayashi, A. Yamada, et al., Isolation of type B influenza virus from the blood of children, Clinical Infectious Diseases 27 (1998) 654 – 655. [18] E.J.C. Claas, M.J.W. Sprenger, G.E.M. Kleter, R. van Beek, W.G.V. Quint, N. Masurel, Type-specific identification of influenza viruses A, B and C by the polymerase chain reaction, Journal of Virological Methods 39 (1992) 1 – 13. [19] D.E. Wentworth, M. McGregor, M.D. Macklin, V. Neumann, V.S. Hinshaw, Transmission of swine influenza virus to humans after exposure to experimentally infected pigs, Journal of Infectious Diseases 175 (1997) 7 – 15.