Reviews in Medical Virology

REVIEW

Rev. Med. Virol. 2002; 12: 375–389. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rmv.370

Molecular diagnosis of influenza Joanna S. Ellis* and Maria C. Zambon Respiratory Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, 61 Colindale Avenue, Colindale, London NW9 5HT, UK

SUMMARY The past decade has seen tremendous developments in molecular diagnostic techniques. In particular, the development of PCR technology has enabled rapid and sensitive viral diagnostic tests to influence patient management. Molecular methods used directly on clinical material have an important role to play in the diagnosis and surveillance of influenza viruses. Molecular diagnostic tests that allow timely and accurate detection of influenza are already implemented in many laboratories. The combination of automated purification of nucleic acids with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. The recent development of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to transform influenza diagnosis. While molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they remain invaluable in assisting our understanding of the epidemiology of influenza viruses. Copyright # 2002 John Wiley & Sons, Ltd. Accepted: 7 August 2002

INTRODUCTION Influenza viruses continue to be a major cause of respiratory tract infection, resulting in significant morbidity, mortality and financial burden. Each year, increased hospitalisation rates and excess deaths are attributable to influenza infections. Although influenza for most people is a mild illness, which is resolved in 1–2 weeks, complications associated with influenza infection can occur in both the upper and lower respiratory tract, some of which may be fatal [1]. The potential for developing complications is higher in certain risk groups, such as the elderly and individuals with chronic medical conditions. An accurate diagnosis of influenza by a physician is difficult since several different pathogens can produce respiratory ill*Corresponding author: Dr J. S. Ellis, Respiratory Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, 61 Colindale Avenue, Colindale, London NW9 5HT, UK. E-mail: [email protected] Abbreviations used BDNA, branched chain DNA; EIA, enzyme immunoassay; HI, haemagglutination inhibition; HMA, heteroduplex mobility assay; IF, immunofluorescence; ILI, influenza-like illness; LCR, ligase chain reaction; M, matrix; NASBA, nucleic acid sequence-based amplification; NP, nucleoprotein; NS, non-structural; PIV, parainfluenza; RFLP, restriction fragment length polymorphism; WHO, World Health Organisation.

Copyright # 2002 John Wiley & Sons, Ltd.

nesses with similar clinical symptoms. Consequently, there is a requirement for sensitive and rapid diagnostic techniques to verify the clinical diagnosis of influenza and improve the quality of surveillance systems. Moreover, the development of specific anti-influenza neuraminidase inhibitors has increased the potential for rapid and accurate diagnostic tests for influenza viruses to contribute to the management of patients. A number of laboratory methods for the diagnosis of influenza are currently available (Table 1). Each of these methods has advantages and disadvantages, and some, or all, of these factors may influence the method of choice. Although molecular technology has transformed the diagnosis of a number of diseases caused by RNA viruses, for example HIV [2], the application of molecular methods to the detection of respiratory pathogens is still comparatively new and expanding rapidly. MOLECULAR METHODS FOR THE DETECTION OF INFLUENZA VIRUSES

Choice of molecular assay A number of molecular methods can be employed for the detection of influenza viruses, the majority of which are based on PCR methodology (Table 2). When selecting which assay to use, there are a

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J. S. Ellis and M. C. Zambon

Table 1. Comparison of the properties of diagnostic methods for influenza virus detection Method Sample required

Cost per test Speeda (£)

Through putb

Advantages

Culture Nasopharyngeal aspirate Nose and throat swab Bronchoalveolar lavage IF Nasopharyngeal aspirate Nose and throat swab Bronchoalveolar lavage

£10–£20

3–7 days

Low

Whole virus Requires measured infectious virus Virus Highly skilled recoverable Time required

£10

2 h–1 day

Medium

Rapid

EIA

Nasopharyngeal £5–£30 aspirate Nose and throat swab Bronchoalveolar lavage RT-PCR Nasopharyngeal £30–£50 aspirate Nose and throat swab Bronchoalveolar lavage Post-mortem tissue Serology Serum £5 HI/CFT

15 min–1 day High

1–2 days

High

2 days

High

Rapid Low skill Can be ‘near patient’ testing

Disadvantages

Requires intact cells Highly skilled No virus recoverable Specialised equipment Cost No virus recoverable

Sensitive Allows further molecular analysis

Cost Highly skilled No virus recoverable Specialised equipment/ laboratory Sensitive and Retrospective specific Paired samples needed

a

Speed with which results are available from the time the specimen is taken. Number of specimens handled in unit time.

b

number of factors that should be taken into consideration. These include the requirement for qualitative, semi-quantitative or quantitative data, and the nature and number of samples to be analysed. In addition, the available time and resources of the laboratory where the work is to be performed and the skill of the staff involved must also be considered.

Hybridisation Molecular hybridisation methods have been used extensively in basic and applied virology because of their technical flexibility and high specificity. Copyright # 2002 John Wiley & Sons, Ltd.

Using these techniques, DNA and RNA viruses have been detected directly in clinical specimens. Influenza A virus RNA has been detected in nasopharyngeal swabs by molecular hybridisation, with a sensitivity of 72% [3]. Hybridisation was more sensitive than both immunofluorescence (IF) and culture, but less sensitive than EIA, which detected influenza virus in 86% of swabs. Molecular hybridisation, however, has not been widely used for the detection of influenza viral RNA in clinical material, largely due to the development and optimisation of more sensitive and less timeconsuming techniques. Rev. Med. Virol. 2002; 12: 375–389.

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377

Table 2. Properties of molecular methods suitable for detection of influenza viruses Method

Advantages

Disadvantages

Hybridisation

High specificity Flexible formats Inexpensive Sensitive and specific Allows further product analysis Sensitive and specific Tests >1 target per assay Allows further product analysis Sensitive and specific High throughput Not limited by product size Can be multiplexed Sensitive and specific Rapid Can be quantitative Can be multiplexed Sensitive and specific Can be quantitative

Sensitivity Time-consuming

PCR Multiplex PCR

PCR-EIA

Real-time PCR

NASBA

Microarrays

Sensitive and specific Tests many targets per assay

PCR The development of PCR analysis in 1985 made possible the diagnosis of virus infection through sensitive detection of specific viral nucleic acids [4]. PCR techniques have been developed for the specific detection and subtyping of influenza viruses to obtain rapid diagnostic results. In these assays, purified influenza viral RNA from culture fluids or clinical specimens is first reverse transcribed to cDNA, by either avian myeloblastosis virus or moloney murine leukaemia virus reverse transcriptase, using random hexanucleotides, universal primer complementary to the 30 end of all influenza vRNAs, or a sequence specific primer. The use of random hexanucleotides or a universal primer, instead of target sequence specific primers has the advantage that the cDNA from both viral genomic RNA and mRNA transcripts is synthesized, thereby increasing the number of target regions that could be amplified by PCR. A single round of amplification may be used and the specificity of the reaction confirmed by hybridisation with product specific probes. Alternatively, Copyright # 2002 John Wiley & Sons, Ltd.

Qualitative, not usually quantitative Requires extensive optimisation to ensure no false negatives or primer competition May be limited by product size Does not allow further product analysis Requires additional evaluation/validation before use Requires specialised equipment Product analysis not always feasible

Three enzymes used Time-consuming RNA product analysis less feasible Requires specialised equipment and extensive development Does not allow further product analysis

nested-primer sets may be utilised to amplify the target region. The choice of gene target is influenced by the prospective application of the assay. For typespecific diagnosis of influenza A, B or C infection, internal genes such as nucleoprotein (NP) and matrix (M) genes are usually chosen, since these are highly conserved within influenza types. Where information on the subtype of influenza A is required, then the genes encoding the surface antigens are targeted. The use of multisegment PCR using primers complementary to the conserved 13 nucleotides at the 50 terminus and 12 nucleotides at the 30 terminus allows the detection of all segments in a single reaction [5]. Moreover, primer sets based on the 15 and 21 nucleotide terminal sequences enable the specific amplification of each of the eight RNA segments and subsequent analysis of all 15 HA and 9 NA subtypes of influenza A virus [6]. Reports on the applications of PCR techniques to the detection of influenza A and B viruses first appeared in the early 1990s. Initial reports described Rev. Med. Virol. 2002; 12: 375–389.

378 the amplification of influenza A H1 nucleic acid from both nasopharyngeal lavages [7] and nasopharyngeal aspirates [8], and influenza A H1, H3 and influenza B HA specific sequences from throat swabs [9,10]. In a study by Zhang and Evans [11] the M genes of influenza A and B viruses and the influenza C HA gene were targets for type-specific nested-primer sets. Subtype-specific primers targeted conserved sequences within the three HA or two NA subtypes of different human influenza isolates. Although the assays were shown to be both sensitive and specific for the typing and subtyping of cultured influenza viruses, their application to the analysis of viruses directly from clinical material was not assessed. Further studies reported the detection of influenza A, B and C viruses in respiratory secretions, using primers targeting specific sequences in the non-structural (NS), or M genes [12–14]. In these initial studies, a small number of respiratory samples were used to evaluate the applicability of the RT-PCR assays for the diagnosis of influenza in clinical specimens. Although early assays used different extraction procedures, gene targets and methods for discrimination of products, the sensitivity of PCR for the detection of influenza viruses was shown to be at least equivalent to that of culture. The majority of RT-PCR assays described to date have utilised primers deduced from the nucleotide sequences of human influenza viral genes. The occurrence of zoonotic events in Hong Kong and Mainland China caused by influenza A H5N1 and H9N2 avian viruses have highlighted the additional requirement for rapid tests to detect viruses originating from non-human hosts. Recently, a single-tube RT-PCR assay for the specific detection of influenza A viruses from multiple species has been reported [15]. A primer set based on conserved regions of the M gene was shown to detect a panel of 25 genetically diverse virus isolates obtained from birds, humans, pigs, horses and seals which included all known subtypes of influenza A virus. To detect and partially characterise influenza A viruses from different animal species, a combined RT-PCR heteroduplex mobility assay (HMA) approach has been described [16]. An M gene-based RT-PCR using nested primer pairs was shown to be specific for the detection of human, avian and swine influenza A viruses, of 15 different subtypes. PCR amplicons were then analysed by HMA to determine the Copyright # 2002 John Wiley & Sons, Ltd.

J. S. Ellis and M. C. Zambon host of origin of the M gene segment. This methodology offers a rapid and sensitive means of screening for novel or unusual influenza viruses. The segmented nature of the genome of influenza A, B and C viruses makes reassortment among viruses an important mechanism for generating genetic diversity. Reassortment between influenza A viruses of different subtypes is of particular importance because of its role in the generation of new pandemic strains in humans. Hence, there is a need for the development of rapid molecular assays that provide subtype information on both the HA and NA of influenza A viruses. This is highlighted by the recent emergence of influenza A H1N2 reassortant viruses in several countries during 2001–2002, following reassortment between circulating influenza A H1N1 and H3N2 viruses [17]. The majority of existing molecular diagnostic tests for influenza A viruses rely on the detection and typing of the HA gene alone, with only a few laboratories undertaking antigenic or genetic analysis of the NA. As yet, PCR assays targeting specific NA subtype sequences have only been described for the N1 and N2 genes of influenza A viruses [11,18].

PCR-EIA A modification of the PCR technique, PCR-enzyme immunoassay (PCR-EIA) has been described [19–22]. A single round RT-PCR is performed and the resulting amplicons identified by hybridisation in solution to a biotinylated RNA probe and detected in an EIA. The potential efficiency of influenza A, M-gene specific PCR-EIA for use in clinical diagnosis was evaluated in a study by Cherian [19]. A total of 90 nasal wash specimens were obtained daily over a 10-day period from nine human volunteers infected with influenza A virus. The PCR-EIA and cell culture had equivalent sensitivities during the first 4 days following infection. However, PCR-EIA was substantially more sensitive than culture later in the course of infection. From days 5 to 9, PCR-EIA could detect influenza A virus RNA in 38% of samples, whereas only 9% were culture positive during that period of time. The results demonstrated that clinical symptoms and shedding of viral RNA might persist after virus becomes undetectable by traditional cultivation methods. The clinical importance of the detection of viral RNA sequences in culture negative samples remains to Rev. Med. Virol. 2002; 12: 375–389.

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379

be established, although increased sensitivity of this analytical method has several advantages, including extending the window for detection of virus and diagnosis of infection.

Multiplex PCR The term multiplex PCR refers to the inclusion of more than one primer set in an amplification reaction, to detect the presence of more than one gene or genome segment in a single pathogen, or in more than one pathogen. Several multiplex PCR assays have been described for the detection of respiratory pathogens in clinical material (Table 3). Multiplex PCR assays have been designed to type and subtype influenza A H1, H3 and influenza B virus in clinical specimens [18,23]. A multiplex PCR assay using nested primer sets targeting conserved regions of the HA genes of influenza A H1, H3 and influenza B viruses has been used in a prospective surveillance of influenza in England during the 1995–1996 winter season [23]. A total of 619 combined nose and throat swabs from patients with an influenza-like illness (ILI) were analysed by cell culture and multiplex PCR. Of these,

39.7% were positive by multiplex PCR, compared to 32.3% that yielded influenza viruses by cell culture. The benefits of multiplex RT-PCR compared with cell culture for detection were most evident during the peak period when influenza virus was circulating in the community. During this period, 38.9% of the samples received were positive by cell culture, whereas 57.5% were positive for influenza virus by multiplex PCR, indicating the improved positive predictive value of multiplex PCR compared with cell culture. Since infection with respiratory pathogen(s) other than influenza viruses can result in clinical symptoms clinically indistinguishable from true influenza, samples negative for influenza virus from patients with ILI may contain these pathogens. The multiplex PCR assay described above has been modified to detect and subtype RSV A and B, in addition to influenza A and B viruses, in a single reaction [24]. In this study, an excellent (100%) correlation between multiplex PCR and the results of influenza culture was demonstrated. The test was also able to detect mixed viral infections in both simulated specimens and clinical samples.

Table 3. Multiplex RT-PCR assays described for detecting human respiratory pathogens Target

Number of pathogens detected

Type Method of of product PCRa identification

Sensitivity

Reference

Influenza A (H1, H3) and B Influenza A and RSV

3

S

25–100 pfu

[18]

2

S

2 pfu

[20]

Influenza A (H1, H3) and B Influenza A (H1, H3), B and C Influenza A (H1, H3), B and RSV A and B Influenza A and B, RSV A and B, HPIV-1, -2 and -3 (Hexaplex assay) Influenza A and B, RSV, HPIV-1, -3, enterovirus, adenovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae

3

N

1–5 pfu

[23]

4

S

Not reported

[49]

5

N

1 pfu

[24]

7

S

5–10 genome copies

[22]

9

S

100 genome copies

[21]

a

Size/Gel electrophoresis Size/Gel electrophoresis Size/Gel electrophoresis Size/Gel electrophoresis Size/Gel electrophoresis Probe/Enzyme hybridisation assay Probe/PCRELISA

Number of rounds of amplification; S, single, one round of amplification, N, nested, two rounds of amplification.

Copyright # 2002 John Wiley & Sons, Ltd.

Rev. Med. Virol. 2002; 12: 375–389.

380 The use of multiplex RT-PCR enzyme hybridisation assays has allowed the detection of a wide range of respiratory pathogens in a single test, since discrimination of the products is not limited by size differences detectable following gel electrophoresis. The Hexaplex assay (Prodesse, Milwaukee) includes primers specific for the M and NS genes of influenza A and B viruses respectively, as well as other primer pairs for the detection and quantification of RSV A and B, human parainfluenza (PIV) type-1, 2 and 3 RNA in clinical specimens [22]. A hot start RT-PCR EIA protocol for the simultaneous detection of nine respiratory pathogens including influenza A and B viruses, PIV-1, PIV-3, RSV, enteroviruses, adenovirus, Chlamydia pneumoniae and Mycoplasma pneumoniae has been developed. This assay has been evaluated using 1118 nasopharyngeal aspirates collected from hospitalised children over a number of influenza seasons [21,25]. Of these, 395 (35%) were positive for one of the nine pathogens. Over the period of the study, 20% of these were positive for influenza A virus and 2.8% were positive for influenza B virus. Seasonal variations in the rates of detection of the different infectious agents were seen. The multiplex RT-PCR showed excellent levels of concordance (98%) for influenza A virus detection with data obtained by commercially available enzyme immunoassay.

NASBA In the past few years, amplification procedures other than PCR have been developed. These include branched chain DNA (bDNA) assay [26], ligase chain reaction (LCR) assays [27], and nucleic acid sequence-based amplification (NASBA). Of these assays, NASBA has been most widely applied for the detection of viral nucleic acid. In the NASBA system amplification is carried out isothermally using three enzymes simultaneously (avian myeloblastosis virus reverse transcriptase, T7 RNA polymerase and RNase H) with two specially designed DNA oligonucleotide primers. The technique is particularly suited to the detection of RNA viruses, since RNA polymerase amplifies RNA without conversion to complementary DNA [28]. NASBA has been applied successfully to a number of RNA templates including HIV-1 [29], HCV [30], rhinoviruses [31] and enteroviruses [32]. Interestingly, a NASBA technique has very recently been developed that allows the detection Copyright # 2002 John Wiley & Sons, Ltd.

J. S. Ellis and M. C. Zambon of avian influenza A subtype H5 isolates of the Eurasian lineage from allantoic fluid harvested from inoculated chick embryos [33]. Both generic primers (for the amplification of both highly pathogenic and low pathogenic H5 HA sequences), and primers specific for pathogenic H5 HA sequences were designed and evaluated for the detection of H5 HA sequences and the differentiation of highly pathogenic H5 sequences from low pathogenic H5 sequences. However, NASBA technology has yet to be applied to the detection of human influenza viruses in respiratory specimens. QUALITATIVE VERSUS QUANTITATIVE ASSAYS The most sensitive molecular techniques use an enzymatic step to amplify target nucleic acid before detection of the specific sequence. Typically, the logarithmic amplification of the target sequence results in the amount of DNA product at the end of the PCR having no correlation to the number of target copies present in the original specimen. The qualitative nature of PCR-based applications limited, in the early stages, the use of this methodology to those conditions where only the presence or absence of a specific target sequence was to be assayed. However, many applications require quantification of the number of target molecules in the original specimen. Both PCR-based and non-PCR strategies, such as realtime PCR and NASBA, respectively, have been adapted for quantitation of nucleic acid products, using either internal or external standards. Quantitative molecular assays have been used primarily to determine the viral load in blood specimens for the diagnosis of HIV, HCV and hepatitis B virus infections [34–36]. Quantitation of CMV DNA molecules is required for successful monitoring of this infection in immunocompromised individuals and during therapy. Where traditional diagnostic methods are insensitive, our understanding of the natural history of viral disease has continued to be improved through the use of molecular techniques. In particular, quantitative PCR-based studies have had an important role in the analysis of the natural history of HIV and HCV infections, and subsequently in the development of effective treatment strategies. During antiviral treatments, quantitative techniques can supply information on both efficacy of therapies and selection of drug-resistant Rev. Med. Virol. 2002; 12: 375–389.

Molecular diagnosis of influenza viral variants in real-time, as demonstrated in HIV-1 infection [37,38]. Although quantitative PCR assays have yet to be widely employed in the diagnosis of influenza infections, and the development of such assays is technically demanding, the potential value of such assays in monitoring efficacy of anti-influenza drug treatments and in the monitoring of immunosuppressed patients is apparent. ANALYSIS OF MOLECULAR PRODUCTS One advantage of PCR-based diagnostic assays is that subsequent sequence information can be

381 determined from the assay products, in a variety of ways (Figure 1). Post-amplification methods for analysing sequence variation in PCR amplicons include restriction fragment length polymorphism analysis (RFLP), HMA and nucleotide sequencing.

PCR-RFLP A combination of RT-PCR and enzyme digestion of HA gene amplicons (PCR-restriction assay) has been used to rapidly differentiate influenza A H3 genetic variants that are antigenically similar [39,40]. This technique may also be utilised to dif-

Figure 1. PCR-based amplification methods and product analysis

Copyright # 2002 John Wiley & Sons, Ltd.

Rev. Med. Virol. 2002; 12: 375–389.

382 ferentiate between vaccine strains and currently circulating strains [38] and to assess the gene composition of candidate vaccine strains [41,42]. Amantadine-resistant influenza A viruses have been detected in nasopharyngeal swabs by PCRRFLP [43]. PCR-restriction assays have most recently been used to rapidly genotype and monitor the internal genes of human influenza H1N1, H3N2 and H5N1 influenza A viruses [44]. A possible drawback of the RFLP technique is that mutations in the nucleotide sequence of the gene of interest may lead to the loss or generation of a restriction site. The high mutability of RNA genomes such as influenza viral genes increases the possibility of this occurring.

Heteroduplex mobility assays Heteroduplex formation between amplicons has been used to subtype viral genomes, to screen for genome variants, to detect mutations and to measure diversity within and between viral genome populations [45]. The initial application of HMA in virology was to subtype HIV-1 [46,47]. This methodology has since been applied to the analysis of many RNA viral genomes, including those of influenza A and B viruses. By combining a HAspecific multiplex RT-PCR with HMA, variant strains of influenza A and B viruses can be differentiated [48,49]. Direct amplification of internal genes from clinical material can also be coupled with HMA to allow rapid species identification of the origin of influenza viruses [16].

Nucleotide sequence analysis Variation among amplified fragments indicated by RFLP or HMA can be confirmed by nucleotide sequencing of derived clones or the PCR product itself. In many laboratories sequence analysis of PCR amplicons is routinely performed, particularly on HA gene products where an association between sequence changes with genetic drift is studied [50,51]. Sequence analysis of influenza gene segments amplified by PCR may also be undertaken to provide information on genetic reassortment between influenza A viruses [52–55]. Antigenic drift in influenza viruses results from the progressive accumulation of mutations, including substitutions, deletions and insertions in the influenza viral genes. These mutations arise due to replication of the influenza virus genome by a viral RNA polymerase that lacks proofreadCopyright # 2002 John Wiley & Sons, Ltd.

J. S. Ellis and M. C. Zambon ing activity. Because of this genetic variation, the target regions in newly emerging strains complementary to the primers or probes used in an assay must be regularly analysed by nucleotide sequencing to check for sequence mismatches between the primer or probe, and target sequences. In the event of sequence mismatches, the primers or probes should be updated in order to avoid false negative assay results. APPLICATIONS OF MOLECULAR METHODS

Diagnosis A limited number of studies have compared laboratory diagnostic techniques for influenza viruses, including RT-PCR, with clinical diagnosis. In a study that aimed to predict influenza infections during epidemics by use of a clinical case definition, combined nasal and pharyngeal swab specimens were collected from 100 patients presenting with ILI of <72 h duration [56]. Patients were aged between 6 and 84 years (mean 39.3 years) and had fever together with two clinical symptoms (headache, cough, sore throat and myalgia). The rate of laboratory-confirmed influenza infection was 72% by cell culture and 79% according to the results of multiplex RT-PCR analysis. Cough and fever were the only factors shown to be significantly associated with a positive PCR test for influenza. Early phase III trials of the anti-neuraminidase drug, zanamivir, in Europe and N. America have also provided evidence of excellent concordance between PCR and classical diagnostic methodology [57]. In these trials, 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 percentage of samples positive for influenza A or B virus, by cell culture, serology or PCR were compared. A total of 791 (77%) of 1033 patients with laboratory results from all three methods were confirmed positive for influenza by one or more test results. For 692 (67%) patients, the results of all three tests agreed. 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 Rev. Med. Virol. 2002; 12: 375–389.

Molecular diagnosis of influenza correlation between duration of illness, but not antibiotic use, or the risk of complications. Although PCR was more sensitive than either culture or paired haemagglutination inhibition (HI) serology, PCR positivity alone was not more likely to be associated with severity of illness, development of complications or antibiotic usage. The results of these studies confirm that RT-PCR provides rapid and accurate diagnosis in an individual patient and is more sensitive than cell culture or the use of paired serology, for detection of cases of influenza in the community.

Surveillance The effectiveness of PCR for enhancing the surveillance and characterisation of circulating influenza strains has been well documented (Table 4). RTPCR has been compared with other methodologies, such as isolation in culture, EIA and IF for the detection of influenza viruses in samples collected during surveillance of influenza virus activity. A number of RT-PCR assays employed in these studies have used single amplification with primers specific for the M gene [13], or NS gene [12] of influenza A and B viruses, or primers defined in NP, NS and HA genes [18], to type and subtype influenza viruses [58,59]. Others have used the nested primer sets described by Zhang and Evans [11] for typing and subtyping of influenza viruses, either in uniplex assays or in a multiplex system [23,60–63]. Although the RT-PCR assays used in these studies differ in their format, the results of the analyses confirmed that RT-PCR was more sensitive than traditional techniques for influenza virus detection in clinical material. The effectiveness of RT-PCR for the detection of influenza viruses was particularly apparent in surveillance systems where the culture of influenza viruses, for a number of reasons, was found to be difficult and suboptimal. In Portugal, a comparison of multiplex RT-PCR for the detection of influenza viruses with culture, EIA and serology was performed over a 7-year surveillance period, from 1992 to 1999 [63]. There was good correlation between the increase of morbidity, total samples taken and the detection of influenza virus by all the methods, although this was less evident for virus isolation and EIA than for RT-PCR or serology. From a total of 1685 throat swabs collected from cases of ILI, more samples were found to be positive by RT-PCR, than by any other method. Copyright # 2002 John Wiley & Sons, Ltd.

383 Moreover, the detection of influenza by RT-PCR occurred earlier than by any other method in all of the years studied, and consistently showed the best relation with epidemic patterns of morbidity registration. The correlation between detection of influenza viruses by RT-PCR and serology was also observed during surveillance of community influenza infection in general practice in Scotland [64]. Influenza virus was detected in 57% of combined nose and throat swabs by RT-PCR, and 61% by serology. The RT-PCR could be performed in 36 h, whereas a serological result required paired sera taken 2 weeks apart. The exact impact of molecular methodology on surveillance is likely to depend upon the sensitivity of laboratory systems already in place. 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, individual laboratories, in each country according to local circumstances [65,66].

Outbreaks Investigations of outbreaks of respiratory illness are often hindered by the inability to culture the infectious agent responsible. Therefore, the use of molecular techniques directly on clinical respiratory specimens is of particular value in the analysis of respiratory outbreaks. RT-PCR has been used to provide rapid identification of influenza virus outbreaks in schools [39] and on cruise ships [67]. In addition, a combination of PCR and postamplification analysis such as RFLP can reveal the relationship of the causative strain to currently circulating viruses and vaccine strains [39].

Tissue diagnosis Molecular methodology is of particular use in the study of post-mortem specimens [68] and may be of great value in the diagnosis of influenza in the central nervous system [69–71]. Furthermore, analysis of other body tissues may help to clarify mechanisms of pathogenesis [72]. 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 A H1N1 strain in archival material. Amplifiable influenza RNA has been recovered from archival paraffin blocks as old as 79 years [73]. Influenza RNA has been detected by RT-PCR in Rev. Med. Virol. 2002; 12: 375–389.

384 both formalin-fixed and frozen tissue samples from victims of the pandemic [74–76]. The full-length sequences of the two predominant surface proteins of the virus, the HA and NA, and the non-structural gene have now been deduced [75–77]. Most recently, influenza RNA has been recovered from preserved specimens from six wild waterfowl that were captured between 1916 and 1919 [78]. From one of these samples a portion of the HA gene (H1 subtype) has been sequenced. Analysis of this, together with sequence information from the human 1918 samples, is being used to determine the origin of the 1918 pandemic virus. RECENT ADVANCES IN MOLECULAR DIAGNOSTIC METHODS

J. S. Ellis and M. C. Zambon enza A and B viruses. The application of real-time PCR methodology to the surveillance of influenza viruses in samples obtained from the community was assessed during two influenza seasons in Germany [80]. The TaqMan PCR was shown to be more sensitive than cell culture, revealing an overall increase during the period studied of approximately 12%. Furthermore, during the period of highest clinical activity, an increase of 26% influenza virus detection by TaqMan PCR was observed. This confirms the findings of earlier studies demonstrating the particular benefits of PCRbased methodologies compared with cell culture for detection, when influenza viruses are circulating in the community [23,60].

Microarray technology Real-time PCR Recent modifications of PCR analysis called ‘realtime’ PCR are a dramatic development in PCRbased technology. In these assays, a fluorescent signal is generated as the PCR takes place. The combination of nucleic acid amplification and signal detection reduces the time required for nucleic acid detection, since post-PCR processing is not required. Fluorogenic PCR-based methods, using TaqMan PCR technology (Applied Biosystems, Foster City, CA), have just been described for the detection and identification of influenza A and B viruses [79,80]. In these assays, primer/probe sets targeting the M gene of influenza A and B viruses [80], or the M gene of influenza A and HA gene of influenza B [79], were designed to differentiate influenza A and B viruses. In the first of these studies, specific primer/probe sets were also selected to identify HA (H1 and H3) and NA (N1 and N2) subtypes [80]. The fluorogenic probes were labelled with both a fluorescent reporter and a quencher dye. After hybridisation of the probe to the target sequence, the Taq DNA polymerase enzyme cleaved the TaqMan probe by means of its 50 –30 nuclease activity thereby separating the reporter and quencher dyes resulting in increased fluorescence. The specificity of the method in both studies was first evaluated on previously characterised reference strains and isolates. An excellent correlation (100%) was demonstrated between the results of typing and subtyping by the TaqMan PCR and antigenic analysis. In addition, both of the fluorogenic assays were shown to be extremely sensitive for the detection of influCopyright # 2002 John Wiley & Sons, Ltd.

Microarray DNA chips containing immobilised oligonucleotide probes or robotically spotted DNAs can be used to rapidly screen for a number of target molecules amplified from clinical material [81,82]. The ability to simultaneously screen for many nucleic acid sequences implies that microarray technology offers great potential as a diagnostic tool. Reports on the application of array-based methods for diagnosis have now started to appear, and a model DNA array has recently been described for the typing and subtyping of human influenza A and B viruses [83]. A series of 26 primer pairs were designed to amplify multiple fragments from influenza A HA (H1, H2, H3), NA (N1, N2) and MP genes, as well as influenza B virus HA, NA and MP genes. Using these, 24 cDNA products generated were cloned and sequenced, prior to reamplification and spotting onto a modified glass support. Cy3- or Cy5labelled fluorescent probes were then hybridised to these target DNAs. Multiplex PCR primers were also designed for the generation of probes to type and subtype influenza A and B viruses. From the results it was concluded that DNA microarray technology could provide a useful supplement to PCR-based diagnostic methods. The application of this methodology to the detection of influenza viral nucleic acid sequences in clinical material will require further analysis. THE FUTURE FOR MOLECULAR DIAGNOSIS The past decade has seen tremendous developments in molecular diagnostic techniques. Future applications of molecular methods may involve Rev. Med. Virol. 2002; 12: 375–389.

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Table 4. Comparison of influenza diagnosis and surveillance by PCR and cell culture Country

Dates

Number of samples

PCR positive (%)

Culture positive (%)

England and Wales Portugal Scotland Germany

1995–2000 1992–1999 1999–2000 1997–1999

3455 1685 168 2545

34.8 43.5 58.0 25.0

23.0 5.0 11.0 16.0

the use of multiple detection systems in which a number of clinically and epidemiologically related pathogens may be detected and characterised in a single test. It should be noted that although PCR is an extremely powerful and versatile assay method, the process can be labour-intensive. More widespread implementation of molecular testing will therefore depend upon automation, enabling molecular assays to enter the routine clinical laboratory. Robotic systems are currently available to automate each stage of the PCR process, but to date only reports of automated systems for the detection of herpes simplex virus DNA, CMV DNA, or HCV RNA have been published [84–85]. Multiplex PCR methodology coupled with developments in microelectronic detection devices offers the exciting possibility of ‘near-patient’ molecular based testing [87]. While RT-PCR for detection of influenza genes is now a well-established method, most assays are qualitative and not quantitative. Quantification may be invaluable for clinical validation of the diagnosis, and quantitative RT-PCR systems are being designed to allow the determination of viral load in infections. Traditional clinical diagnostic methods require standardisation, quality assurance and quality control measures to be put into place, in order to validate assay results. Due to the extreme sensitivity of PCR assays and similar tests, additional quality control measures must be implemented. Furthermore, nucleic acid detection assays usually involve several processes, and at each stage potential sources of error may occur (Table 5). Human error can occur at all stages, but can be reduced by automation of some, or all of the steps involved. Validation of all reagents should be performed prior to their use in the assay, since errors due to reagent failures can occur throughout the procedure. The integrity of both positive and negative results can be established by the incorporation Copyright # 2002 John Wiley & Sons, Ltd.

of appropriate positive and negative controls, which are processed in an identical manner to the test samples in the assay procedure. The detection and characterisation of influenza isolates and the identification of newly emerging variants, is the foundation of the World Health Organization (WHO) influenza global surveillance network. Information on the antigenic properties of circulating influenza viruses compared with reference and vaccine strains is important for the formulation of influenza virus vaccines. For this reason, optimised influenza surveillance requires both sensitive detection of influenza strains and isolation, followed by full characterisation of the antigenic properties of virus isolates. Although

Table 5. Sources of potential error in influenza viral nucleic acid detection methods Procedure

Potential outcome

Extraction of nucleic acid RNA/DNA degraded Inhibitors present Incomplete extraction Contamination introduced Reverse transcription Transcription errors Enzyme failure Amplification Contamination Inhibition Insensitivity Non-specificity Enzyme failure Detection Non-specificity Enzyme failure

False False False False

negative negative negative positive

False negative False negative False positive False negative False negative False negative/ positive False negative False negative/ positive False negative

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