Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR

Journal of Virological Methods 117 (2004) 103–112

Rapid detection and simultaneous subtype differentiationof influenza A viruses by real time PCR

Belinda Stonea, Julie Burrowsa, Sonia Schepetiuka, Geoff Higginsa,Alan Hampsonb, Robert Shawb, TuckWeng Koka,∗

a Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, P.O. Box 14, Frome Road, South Australia,Rundle Mall, S.A. 5000, Australia

b W.H.O. Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia

Received 18 September 2003; received in revised form 5 December 2003; accepted 8 December 2003

Abstract

A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhancedvirus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridizationprobes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenzaA H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when itwas culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity,specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively theRT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was completeconcordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.© 2004 Published by Elsevier B.V.

Keywords: Influenza A; RT-PCR; Hybridization probe; Melting curve; Enzyme immunoassay (EIA); Culture; Haemagglutination inhibition (HI); Subtypedifferentiation

1. Introduction

Human influenza is a highly contagious acute respiratorytract disease which can cause severe morbidity and mortality,in particular with elderly or immuno-compromised patients(Glezen, 1982). There are three genera of human influenzaviruses viz. A, B and C. Infection with influenza A virus isthe most severe, with several notable pandemics during thepast century (Potter, 1998). Influenza A viruses are classifiedinto subtypes according to the antigenic composition of theirhemagglutinin (HA) and neuraminidase (NA) glycoproteinson the viral envelope (Wright and Webster, 2001).

The available NA inhibiting drugs, viz. Zanamivir andOseltamivir, for influenza A and B viruses have attractedmore interest than the earlier amantadine and rimantidinecompounds which are effective against influenza A only.

∗ Corresponding author. Tel.:+61-8-82223545; fax:+61-8-82223545.E-mail address: [email protected] (T. Kok).

These antiviral drugs reduce the duration of illness by 1–1.5days when administered within 48 h from onset of symptoms(Gravenstein and Davidson, 2002; Gubareva et al., 2000).However, vaccination remains the primary measure to reducethe frequency of influenza.

The symptoms and signs of influenza infection may besimilar to other types of respiratory viral, chlamydiae orMy-coplasma pneumoniae infections. Prompt diagnosis of in-fluenza infection would facilitate effective patient manage-ment, public health and vaccination programs as well as ap-propriate use of antiviral therapy. The mainstay for rapidlaboratory diagnosis of influenza infection has been the im-munofluorescence test (Gardner and McQuillin, 1974) orenzyme immunoassay (EIA) (Kok et al., 1994; Sarkkinenet al., 1981). The recent availability of real time polymerasechain reaction (PCR) (e.g. Applied Biosystems Taqman andRoche LightCycler) which combines amplification and si-multaneous detection of specific nucleic acid sequences hasimproved rapid detection of viral nucleic acid sequences inspecimens (Burrows et al., 2002; van Elden et al., 2001).

0166-0934/$ – see front matter © 2004 Published by Elsevier B.V.doi:10.1016/j.jviromet.2003.12.005

104 B. Stone et al. / Journal of Virological Methods 117 (2004) 103–112

This study compares a rapid, real time reverse transcrip-tion PCR (LightCycler, Roche Molecular Diagnostics) withdetection by antigen EIA (AgEIA) and enhanced culturefor detection and simultaneous subtype differentiation of in-fluenza A viruses in stored and fresh clinical samples.

2. Materials and methods

2.1. Patients and specimens

There were two groups of specimens tested for influenzaA by AgEIA, enhanced culture or real time reverse tran-scription PCR (RT-PCR). The first (‘stored’) was a group of133 stored (−70◦C) respiratory specimens which included85 nasopharyngeal aspirates, 18 throat swabs, three bron-chioalveolar lavage and 27 sputa. There were 112 specimensthat were AgEIA and/or culture positive for influenza A.The remaining 21 specimens in this group were influenza Anegative and otherwise randomly selected. These were col-lected during the period between January 2000 and Decem-ber 2001, from patients whose ages ranged from 1 monthto 88 years, with a median age of 13 years, average 24years. The specimens were initially stored at 4◦C for 1–3days prior to storage at−20◦C (approximately 1 week) andthen subsequently at−70◦C until retrieved for influenza ART-PCR test. All 133 specimens were tested on receipt inthe laboratory by routine screening with AgEIA and/or en-hanced culture, according to the test request. AgEIA testingwas not requested in 38/133 specimens. The 38 specimenswere tested by enhanced culture only.

The second group (‘fresh’) consisted of 58 respiratoryspecimens: one bronchioalveolar lavage, 45 nasopharyngealaspirates, nine sputa and three throat swabs. These were col-lected in June and December 2002 and were tested uponreceipt by all three methods viz. real time reverse transcrip-tion PCR, antigen EIA and enhanced culture. The age of thepatients who provided the latter specimens ranged from 2weeks to 95 years, with a median age of 63 years, average56 years. In this group of ‘fresh’ specimens there were 33specimens obtained during a local outbreak of influenza (inJune 2002) in a high dependency hospital. The remainingspecimens were randomly selected from routine specimens.

The specimens in both groups were received in 2 ml ofviral transport medium (VTM) which contained base cellculture medium (Dulbeccos Minimal Essential Medium,DMEM) with 1% foetal bovine serum, 16�g/ml of gen-tamicin and 12�g/ml of penicillin. The specimens weretested for influenza A and B, respiratory syncytial virus,adenovirus, parainfluenza 1, 2 and 3 viruses andM. pneu-moniae by rapid antigen enzyme immunoassay (AgEIA) asdescribed previously (Kok et al., 1994) or inoculated intomicrowell cell cultures for isolation of the above viruses aswell as other viruses viz. rhinoviruses/enteroviruses at thetime of receipt in the laboratory (Lennette and Schmidt,1979; Schepetiuk and Kok, 1993). Influenza A or B virus

isolates from cell cultures were forwarded to the WorldHealth Organization (WHO) Collaborating Centre for Ref-erence and Research on Influenza, Melbourne, Australia.These isolates were subtyped at the WHO laboratory withspecific antisera by haemagglutination inhibition test.

2.2. Isolation of RNA

Total RNA was extracted from specimens or cell cul-ture fluids using the RNeasy mini kit with QIAshredderaccording to the manufacturer’s instructions (Qiagen, Ger-many). The sample volume used for each extraction rangedfrom 50–350�l (depending on availability) and the extractedRNA was resuspended in 30�l of sterile, distilled water asdescribed previously (Kok et al., 2000).

2.3. Virus amplification primers and hybridization probes

The matrix gene was selected for RT-PCR amplificationand detection of influenza A viral RNA. This gene wasshown to be highly conserved amongst influenza A H1N1,H3N2, H1N2, H2N2, H5N1 and H9N2 by nucleotide se-quence alignments with pairwise distance analysis whichshowed >80% homology between all strains. These humanstrains have been associated with pandemic influenza, localoutbreaks or are the predominant circulating strains (H1N1and H3N2) (Cox and Subbarao, 2000). A selected region ofthe matrix gene alignment for the six strains of influenza Aviruses are shown inFig. 1, together with the amplificationprimer binding, hybridization sensor (acceptor) and anchor(donor) probe sequences.

The PCR primers (HPLC purified) and fluorophore-labelled hybridization probes were obtained from TIBMOLBIOL, Berlin, Germany. The primers used for ampli-fication of influenza A matrix gene were AMPfor1: 5′GACCAA TCC TGT CAC CTC TGA3′ and AMPrev1: 5′GTATAT GAG GCC CAT RCA ACT G3′. The primers cor-responded to nucleotide positions 106–127 and 356–335,respectively, in the influenza A/Taiwan/511/96 (H1N1) se-quence obtained from GenBank accession AF138708. Thehybridization probe sequences and nucleotide positionswere FLU A sensor: LC Red 705-5′GCG TGA ACA CAAATC CTA AAA TCC Cp3′ (132–156) and FLU A anchor:5′TCC TCG CTC ACT GGG CAC GGT G3′-fluorescein(158–180). The PCR product size was 251 nucleotides.Sequence specific hybridization probes were designed todiscriminate between H1N1 and H3N2 strains based ontwo nucleotide mismatches in the corresponding sensor(acceptor) region.

2.4. Reverse transcription PCR (RT-PCR)

Extracted RNA in a volume of 1�l was added to the re-action mix (final volume 20�l) in a LightCycler capillarytube containing 4�l of 5X RT-PCR Reaction Hybridiza-tion Probe mixture (Roche Molecular Diagnostics), 0.4�l

B. Stone et al. / Journal of Virological Methods 117 (2004) 103–112 105

Fig. 1. Alignment of matrix gene from six influenza A virus strains showing the sequence binding regions of the amplification primers (AMP) andhybridization (anchor/sensor) probes. The sensor hybridization probe is homologous to influenza A H1N1 and sequence mismatches with other strainsare located within this region.

of an enzyme mix containing reverse transcriptase and DNApolymerase, 10 U ribonuclease inhibitor (Promega), 4 mMof MgCl2 (final concentration), 0.5�M of AMP rev1 primer,1.0�M of AMP for1 primer, 0.2�M of the 3′FLU-anchorprobe and 0.3�M of the 5′LC705-sensor probe.

The RT-PCR conditions consisted of 1 cycle of reversetranscription at 55◦C for 10 min, followed by one cycle of95◦C for 30 s to denature DNA strands; then 15 cycles of95◦C, 5 s; 62–50◦C, 10 s and 72◦C, 12 s. A touchdownprotocol was used for the annealing step during the first 15cycles. This involved a step-wise decrease of 0.8◦C from aninitial annealing temperature of 62◦C over 15 cycles until afinal temperature of 50◦C was obtained. This was followedby a further 30 cycles of 95◦C, 5 s; 50◦C, 10 s and 72◦C,12 s. A positive (influenza A RNA) and a negative control(water) were included in each test.

2.5. Real time PCR analysis (Roche LightCycler)

Fluorescence values from amplification products formedduring the PCR were measured in the LightCycler everycycle at 705 nm. The melting curve was generated using theLightCycler software program with initial denaturation ofamplified products at 95◦C for 20 s followed by 45◦C/30 sand then increasing the temperature to 85◦C in incrementsof 0.1◦C/s. Fluorescence was continuously measured at705 nm. The reaction chamber was then cooled to 40◦C for30 s prior to opening the chamber to remove the capillarytubes, as recommended by the manufacturer. The melting

curve allows determination of the point at which 50% ofthe probes have denatured from the PCR product. Thispoint is dependant on the length and sequence homologybetween the probe and PCR product and is characteristicfor each of the subtypes. The sensor probe is homologousto H1N1. There are two nucleotide mismatches betweenthe sequence of H1N1 and H3N2 within the region wherethe sensor probe binds (seeFig. 1). The two nucleotidemismatches with H3N2 yielded a lower melting tem-perature (60◦C) between the probe and target sequencecompared to the homologous sequence H1N1 which dena-tured at 65◦C, thus allowing for differentiation of the twosubtypes.

2.6. Preparation of influenza A RNA from plasmid clone

Influenza A RNA transcripts, corresponding to the am-plified matrix gene (seeSection 2.3) were produced fromcloned plasmids prepared from H1N1 A/Taiwan/511/96-likeand H3N2 A/Port Chalmers/1/73-like influenza viruses.The amplified product was purified using QIAquick PCRpurification kit (Qiagen) and cloned into the pGEM®-TEasy vector (Promega) according to the manufacturers’ in-structions. The plasmids were extracted and purified usingQIAprep Spin Miniprep kit (Qiagen). The amplified productwas confirmed by sequence analysis using the pUC/M13primer (Promega). The cloned plasmids were digestedand RNA transcripts prepared using the Riboprobe® invitro Transcription System according to the manufacturer’s


instructions (Promega). The RNA was purified using RNeasyMini Kit (Qiagen). The yield and purity (OD260/OD280 >1.9) were estimated in an Ultrospec 3000 spectrophotometer(Pharmacia Biotech).

2.7. PCR amplification and nucleotide sequence analysesof influenza A haemagglutinin

The haemagglutinin genes of influenza A isolates fromcell culture fluids were amplified using the TitanTM one tubeRT-PCR system (Roche Molecular Diagnostics) in whichboth the reverse transcription and PCR steps were reacted inthe same tube. The primer sequences for amplification andsequence analysis of HA1 gene were provided by K. Sub-barao, CDC, Atlanta (personal communication). The primersequences for amplification of HA1 domain were H1F-6:5′AAG CAG GGG AAA ATA AAA3 ′ and H1R-1193:5′GTA ATC CCG TTA ATG GCA3′ for H1N1, with apredicted length of 1187 bp. The amplification primers forHA1 domain of H3N2 subtype were H3F-7: 5′ACT ATCATT GCT TTG AGC3′ and H3R-1184: 5′ATG GCT GCTTGA GTG CTT3′ with a predicted length of 1177 bp. Thefinal concentrations of the reagents were 200�M dNTPs(Promega), 0.4�M sense and antisense primers, 5 mM DTT,20 U RNasin ribonuclease inhibitor (Promega), 25�l of 5XTitan reaction buffer containing 1.5�M MgCl2, 1�l of Ti-tan High Fidelity enzyme mix (Taq DNA polymerase, PwoDNA polymerase and avian myeloblastosis virus (AMV)reverse transcriptase) and 3�l of template RNA. The reac-tion volume was brought up to 50�l with deionised, sterilewater.

Viral RNA was reverse transcribed at 50◦C for 30 min.The cDNA was denatured for 5 min at 95◦C followed by40 amplification cycles of 95◦C, 30 s and 52◦C, 30 s witha final extension step at 72◦C, 10 min. The amplicons werepurified using a QIAquick PCR purification kit (Qiagen) ac-cording to the manufacturer’s instructions and the amountestimated using an Ultrospec 3000 spectrophotometer (Phar-macia Biotech).

The amplified products were then sequenced by over-lapping regions using two primers binding to the antisensestrand and four primers binding to the sense strand (seeTable 1). The purified viral cDNA (200 ng) was added to aPCR reaction tube containing 4�l of Dye Terminator ReadyReaction Mix version 3, 4 pmol of appropriate sequencingprimer and deionised sterile water added to 20�l. The reac-tion was amplified over 35 cycles of 96◦C, 10 s; 50◦C, 10 sand 60◦C, 4 min, then held at 4◦C in a Corbett Researchthermocycler. The nucleotide sequence was analyzed in anautomated ABI PRISM 3700 DNA Analyser (Perkin ElmerApplied Biosystems). Contiguous regions of the amplifiedinfluenza A haemagglutinin sequence were constructed us-ing the Seqman computer program Lasergene (DNAstar,Madison, WI). These sequences were aligned and analyzedusing MegAlign, Lasergene.

Table 1Primers for sequencing influenza A HA1 gene

Name Sequence 5′–3′ InfluenzaA type

H1R-1110 CCA TCC ATC TAT CAT TCC H1N1H1R-810 TAG ATT TCC ATT TGC CTCH1R-523 CCG TCA GCC ATA GCA AATH1R-365 TTC CTC ATA CTC GGC GAAH1F-272 AAT CAT GGT CCT ACA TTGH1F-481 TTT TAC AGA AAT TTG CTA

H3R-1073 CCT GCG ATT GCG CCT AAT H3N2H3R-792 CAG TAT GTC TCC CGG TTTH3R-570 TGG CAT AGT CAC GTT CAGH3R-362 TAA GGG TAA CAG TTG CTGH3F-282 CAG CAA CTG TTA CCCH3F-490 CTG AAC GTG ACT ATG CCA

2.8. Virus strains

The stock strains and the haemagglutination HA titreof the influenza A viruses used as controls were as fol-lows: influenza A H1N1, A/Taiwan/96/511-like (GenBankaccession AF138708) (HA titre 512); influenza A H3N2A/Port Chalmers/1/73-like (GenBank accession X08092)(HA titre 512). The viruses were grown in MDCK cellstreated with trypsin (1 mg/ml)-DMEM as described pre-viously (Schepetiuk and Kok, 1993). The haemagglu-tinin gene sequence of influenza A viruses were con-firmed by comparison with the GenBank database. TheHA titres were obtained by titrations of the virus stockswith 0.4% guinea pig erythrocytes (Lennette and Schmidt,1979).

2.9. Antigen enzyme immunoassay (AgEIA)

The AgEIA used has previously been described (Koket al., 1994). Briefly, an aliquot (1 ml) of respiratory spec-imen in viral transport medium (VTM) was frozen andthawed once followed by sonication in a Branson Sonicatorfor 20 min at room temperature. The specimen was thenmixed well in a Vortex Mixer and inoculated (50�l/well)into a pair of microwells coated with rabbit antisera (im-munoglobulin fraction) to influenza or other virus. Theinoculated wells were incubated overnight at room temper-ature (ca. 22◦C). This was followed by five washes withPBS/T (0.05% Tween-20) to remove unreacted specimens.Guinea-pig anti-influenza A immunoglobulins (Ig) werethen added (100�l/well) followed by incubation at 37◦Cfor 1 h. Unreacted reagents were then removed by fivewashes with PBS/T (0.05% Tween-20). Rabbit anti-guineapig IgG conjugated with horse radish peroxidase (Dako,Australia) were then added (100�l/well) followed by in-cubation at 37◦C for 1 h. The chromogen and substrateused were 3,3′,5,5′-tetramethylbenzidine dihydrochlorideand 0.03% sodium perborate (Sigma), respectively for theenzyme reaction.


2.10. Enhanced influenza A virus culture

The enhanced virus culture method has previously beendescribed (Schepetiuk and Kok, 1993). In brief, specimens,in viral transport medium, were inoculated (100�l /well)into microwell cultures of Madin Darby Canine Kidney ep-ithelial cells containing 1 ml of DMEM (without serum)with trypsin (1�g/ml), penicillin (12�g/ml) and gentam-icin (16�g/ml) (Schepetiuk and Kok, 1993). The microwellswere then centrifuged at 1000 g/1 h/35◦C and the mediumreplaced with 1 ml of fresh DMEM containing trypsin andantibiotics. The cultures were incubated for 6 days, followedby one freeze/thaw cycle and the cell culture fluid weretested for presence of influenza A virus in the AgEIA.

2.11. Statistical analysis

A specimen was considered as a ‘true’ positive for in-fluenza A when it was culture positive or both of the othermethods (AgEIA and matrix gene RT-PCR) yielded a posi-tive test result. The sensitivity of a test was calculated to bethe proportion of positive specimens amongst those that wereconsidered as ‘true’ positives. The test specificity was cal-culated to be the proportion of negative specimens amongstthose that were culture negative or with two other tests neg-ative. The positive predictive value (PPV) of the test was theproportion of specimens that was culture or one other testpositive, when the test was positive. The negative predictivevalue (NPV) was calculated using similar criteria for speci-mens which were negative in the test. If a specimen was nottested by AgEIA (as it was not requested by the physician)or yielded bacterial contamination in culture, this was notincluded in the statistical analysis of the assay. Statisticalanalysis was calculated by categorical modeling to comparedifferences in proportions between the three methods.

3. Results

3.1. Sensitivity of RT-PCR

The detection sensitivity of influenza A RNA by RT-PCRwas calibrated with in vitro generated influenza A H1N1 andH3N2 RNA transcripts. Ten fold falling dilutions of RNAwere titrated and tested in the RT-PCR with hybridizationprobes. The limit of detection per reaction (20�l) for H1N1was determined to be 120 copies with the correspondingprobe and target RNA hybrid melting temperature of 65◦C.The detection limit with H3N2 was 350 copies with meltingtemperature of 60◦C. No fluorescence signal was detectedwith lower amounts of the corresponding RNA transcripts.

The detection sensitivity of RT-PCR was also titratedagainst HA units of the H1N1 and H3N2 virus stocks. Bothvirus stocks yielded endpoint haemagglutination dilutionsof 1/512. The endpoint dilutions by RT-PCR for H1N1 andH3N2 viruses were 1/1,250,000 and 1/6,250,000 respec-

tively. These corresponded to 4.1 × 10−4 and 8.2 × 10−5

HA units, respectively.

3.2. Specificity of RT-PCR

The specificity of the RT-PCR was confirmed with spec-imens and viral isolates in cell cultures that were identi-fied previously by rapid AgEIA or virus culture for otherviruses/agent. Specimens or cell culture fluids which werepositive for influenza B, respiratory syncytial virus, parain-fluenza 1, 2 and 3 viruses, adenovirus orM. pneumoniaewere negative in the real time RT-PCR for Influenza A.Fig. 2shows the fluorescence signals obtained in the Light-Cycler with hybridization probe detection to influenza AH1N1, H3N2, an equal volume mix of H1N1 and H3N2and uninfected MDCK cell cultures. The melting tempera-ture of the target and probe hybrid observed for H1N1 was65◦C and for H3N2 was 60◦C. In the sample that containeda mix of H1N1 and H3N2 there were two melting temper-ature values obtained, as expected, corresponding to eachsubtype.

3.3. Detection of influenza A virus by rapid AgEIA,enhanced culture and RT-PCR in stored and prospectivelycollected fresh specimens

3.3.1. Stored specimensTable 2ashows the results of the 133 stored specimens

were tested by enhanced culture and RT-PCR for influenzaA. There were 95 specimens test by rapid AgEIA. Therewas one specimen that yielded bacterial contamination andthis was excluded from the analysis of the enhanced cul-ture. There were 113 specimens that were reactive in oneor more of the influenza A tests, with 53 positive by rapidAgEIA, 104 by culture and 93 by RT-PCR. The criteria fora true positive influenza A were fulfilled in 111/133 spec-imens. The sensitivity, specificity, PPV and NPV of eachtest are shown inTable 2a. The variance in sensitivity be-tween each of the three tests was shown to be significant(P = 0.0007,χ2 14.65, degrees of freedom 2). The speci-ficity or PPV variance between the tests was not consideredsignificant. However, the NPV of 78.6% for culture was sig-nificantly different from either AgEIA or RT-PCR, with thelatter two tests showing no significant variance (P = 0.64,χ2 0.22).

There were 104 specimens that yielded influenza A byenhanced culture. There were 63 samples that were sub-typed by HI as influenza A/New Caledonia H1N1 (23/63),A/Bayern H1N1 (7/63) or A/Moscow H3N2 (33/63). Theremaining 41 samples did not yield sufficient virus for HIsubtyping. The sample that was influenza A positive by thematrix gene RT-PCR (presumptive H3N2), but negative inthe other two tests was also positive when tested by the HA1gene RT-PCR. Nucleotide sequence analysis of the HA1gene from this specimen corresponded with H3N2 subtype.


Fig. 2. Quantification curves relating cycle number (a) or melting temperature curves (b) and hybridization probe fluorescence signals obtained in theLightCycler during real time PCR amplification of influenza A matrix gene with H1N1, H3N2, an equal mix of H1N1 and H3N2, uninfected MDCKcells and sterile water.

Randomly selected influenza A positive cell culture fluids(enhanced culture) from 32 specimens were amplified withthe influenza A HA1 primers and their sequences analyzed.There were 15 isolates identified as H1N1 and 17 as H3N2.All 32 samples were positive by the matrix gene RT-PCRand there was complete concordance between the presump-tive subtype identification by matrix gene RT-PCR (basedon specific melting temperature in the LightCycler and thesubtype assigned) and HA1 gene sequence analysis. Therewas complete concordance between HI subtype results andHA1 gene sequence analysis with 27/32 samples. There were5/32 samples that did not yield sufficient virus for HI sub-typing. The phylogenetic comparisons of these samples areshown with their HI subtypes inFig. 3.

3.3.2. Fresh specimensIn this group of 58 specimens, all were tested (prior to

storage) by the three tests on receipt in the laboratory. Therewere 29 specimens that were reactive in one or more of theinfluenza A tests—15 positive by AgEIA, 23 by culture and29 by RT-PCR (Table 2b). All of the 29 influenza A reactivespecimens were from the hospital outbreak and showed sim-ilar melting temperature curves to the H3N2 positive controlin the LightCycler. There were 24 specimens in which the re-sults met the case definition of a true positive influenza. Twospecimens yielded bacterial contamination in the enhancedculture and were excluded from the statistical analysis ofculture results. The sensitivities of AgEIA, enhanced cultureand RT-PCR were 62.5, 100 and 100%, respectively (see


Table 2Detection of influenza A virus by AgEIA, enhanced culture and RT-PCR in 133 stored specimens (a) and 58 prospectively collected specimens (fresh) (b)

n AgEIA Culture RT-PCR χ2 P

(a) Stored samples45a + + +31 N/T + +9b − + +6c + − +1 + contam. +

12d − + −7 N/T + −1 + − −1 − − +

20e − − −(Total) 133

Sensitivity 71.2% (52/73) 94.5% (104/110) 82.9% (92/111) 14.65 0.0007Specificity 95.5% (21/22) 100.0% (22/22) 95.5% (21/22) 2.19 0.3345PPV 98.1% (52/53) 100.0% (104/104) 98.9% (92/93) 2 0.3673NPV 50.0% (21/42) 78.6% (22/28) 52.5% (21/40) 10.16 0.0062

(b) Fresh samples14 + + +9 − + +1 + contam. +4 − − +1 − contam. +

29f − − −(Total) 58

Sensitivity 62.5% (15/24) 100.0% (23/23) 100.0% (24/24) 14.4 0.0007Specificity 100.0% (33/33) 100.0% (33/33) 87.9% (29/33) 4.55 0.1027PPV 100.0% (15/15) 100.0% (23/23) 82.8% (24/29) 6.04 0.0488NPV 78.6% (33/42) 100.0% (33/33) 100.0% (29/29) 11.45 0.0033

The number of specimens tested per row is shown in column ‘n’. Statistical analysis are shown asχ2 (chi-squared) andP values calculated with 2degrees of freedom. PPV and NPV are positive and negative predictive values, respectively; ‘contam.’ refers to inoculated cell cultures which yieldedbacterial contamination.

a 1/45 Parainfluenza 3 AgEIA and enhanced culture positive, 1/45 Adenovirus culture positive.b 1/9 Parainfluenza 3 AgEIA positive.c 1/6 Parainfluenza 3 culture positive.d 1/12 Parainfluenza 3 AgEIA positive.e 2/20 RSV culture positive, 2/20 Parainfluenza 1 culture positive, 1/20 Parainfluenza 3 culture positive and 1/20 Parainfluenza 3 AgEIA and culture

positive.f 1/29 RSV AgEIA positive; 2/29 Parainfluenza 3 culture positive.

Table 2b). The variance of sensitivities between AgEIA andeither of culture or RT-PCR was significant (P = 0.0007,χ2 14.40, degrees of freedom 2). However, the variance be-tween the test specificity was not significant (P = 0.1027with χ2 4.55, degrees of freedom 2 (seeTable 2b)). ThePPV variance between RT-PCR and AgEIA or culture wassignificant. The NPV of 78.6% for AgEIA and 100% forculture or RT-PCR showed significant variance.

In the group of 23 specimens which were influenzaA culture positive, 19 were subtyped by HI as influenzaA/Moscow/10/99-like (H3N2). The remaining four speci-mens did not yield sufficient virus for HI subtyping. Therewere five specimens obtained from the outbreak that werematrix gene RT-PCR positive but were negative by AgEIAor culture (one was contaminated). However all five spec-imens were also HA1 gene PCR reactive and sequenceanalysis showed that all corresponded with H3N2 subtype.

4. Discussion

This study describes the development of a real timeRT-PCR for the influenza A matrix gene and its evalua-tion by comparison to AgEIA and enhanced culture fordetection of influenza A virus in two groups of respiratoryspecimens—stored and fresh samples. The RT-PCR showedsensitivity of 350 copies per reaction of H3N2 in vitro tran-scribed RNA and 120 copies per reaction of H1N1 RNArepresenting the influenza A types in common circulationat the time of the study. Although not formally tested, wewould expect the primers and most likely the probes to be re-active with H1N2, H2N2, H5N1 and H9N1 strains (Fig. 1).Some detection limits for influenza A virus matrix genereported recently by TaqMan-PCR were 0.1 TCID50/ml(Schweiger et al., 2000), 0.2 TCID50/ml (Fouchier et al.,2000) and 0.006–0.02 TCID50/ml (van Elden et al., 2001)


Fig. 3. Phylogenetic comparison of influenza A H1N1 (a) and H3N2 (b) isolates, from stored samples in this study, by nucleotide sequence analysis ofHA1 gene with the corresponding subtype identification by haemagglutination inhibition. The phylogenetic tree was deduced by unweighted pair groupmethod with arithmetic mean (UPGMA) algorithm using the Kodon Applied Maths version 1 (2002) software package. The scale represents numberof base changes per hundred nucleotides. Influenza A/New Caledonia/20/99 (H1N1-like); A/Beijing/26/95 (H1N1-like); A/Moscow 10/99 (H3N2-like)and A/Sydney/5/97 (H3N2-like) have been included in previous vaccine composition [Macken, 2001 #419]. Influenza A/Taiwan/1/86 (H1N1-like);A/Wuhan/359/95 (H3N2-like) and A/Shanghai/24/90 (H3N2-like) were included for comparison purposes. (’NotRecovd’ denotes HI subtype notavailable).

or 0.01–0.1 TCID50/ml by LightCycler (Poddar, 2003).The different plating efficiencies of various assays, as wellas with different virus strains, do not permit direct com-parisons. However, it is acknowledged that TCID50 is anestimate of the amount of viable virus. A virus stock cul-ture would be expected to contain specific genomic nucleicacids from viable as well as non-viable virus. Previousstudies have shown amounts of 100 TCID50/ml of influenzaA virus (Doller et al., 1992) or 1000 TCID50/ml influenza

A (H3N2) virus (Douglas, 1975; Knight et al., 1969) ofrespiratory secretion in patients with influenza infection. Ifwe take an estimate of one TCID50/ml of influenza virusto contain approximately 100 copies of viral RNA (Whiteand Fenner, 1986), the latter quantitation studies would beexpected to show the equivalent of 104–105 copies of viralRNA/ml of secretion. The reported matrix gene sensitivi-ties are therefore similar to the sensitivity of our assay asestimated by in vitro transcribed RNA.


In this study, we used the criteria of culture positive orat least two test methods positive to define a ‘true’ in-fluenza positive specimen. In the group of stored specimens,the culture sensitivity of 94.5% was the highest followedby RT-PCR (82.9%) and AgEIA (71.2%). The three testmethods yielded similar specificity and PPV, all were >95or >98%, respectively. These specimens were tested in theAgEIA and/or in the enhanced culture upon receipt in thelaboratory, but were tested by RT-PCR after storage for ap-proximately 2 years. In contrast, the second group of speci-mens (‘fresh’) was tested by all three methods upon receiptin the laboratory without prior storage. In these specimens,enhanced culture and RT-PCR each showed 100% sensitiv-ity and AgEIA a sensitivity of 62.5%, but the specificity ofthe tests (RT-PCR, 87.9%; AgEIA, 100% and enhanced cul-ture, 100%) did not differ statistically. The positive samplesin this group were all derived from an outbreak of influenzaA in a high dependency chronic care hospital. (A detaileddescription of this outbreak and vaccination status of thepatients will be provided in a subsequent report).

The lower sensitivity of RT-PCR with specimens from the‘stored’ group, compared to those in the ‘fresh’ group waslikely due to RNA degradation from long term storage of thespecimens for up to three days at 4◦C, then at−20◦C forca. 1 week and subsequently for a period of more than 1 yearat−70◦C, prior to testing for influenza A viral RNA. Whenthe extracted RNA from these specimens were separatedby electrophoresis in a RNA gel (Sambrook et al., 1989),gross degradation of RNA were observed as judged by theabsence of 18S and 28S ribosomal RNA (data not shown).The RNA degradation would likely have contributed to neg-ative RT-PCR results as well as the low NPV of 52.5% inthe specimens from the ‘stored’ group. The mechanism fordegradation of viral RNA may parallel that of mRNA frommammalian cells (van Hoof and Parker, 2002). It is likelythat had matrix gene RT-PCR analysis of these ‘stored’ spec-imens been tested at the time they were collected initially ahigher sensitivity would have been obtained.

In this study there was one specimen in the ‘stored’ group(Table 2a) and five specimens (Table 2b) that were matrixgene RT-PCR positive but negative by AgEIA and culture orcontaminated. Each of these samples yielded a typical H3N2melt curve in the matrix gene RT-PCR and sequence anal-ysis of the HA1 gene corresponded with influenza A H3N2subtype. If these samples are considered as true positives,then the resolved analysis for RT-PCR sensitivity, speci-ficity, PPV and NPV would be 83.0 (93/112), 100 (21/21),100 (93/93) and 52.5% (21/40), respectively in the group ofstored specimens and 100 (24/24), 100 (29/29), 96.6 (28/29)and 100% (29/29), respectively in the group of fresh speci-mens.

The AgEIA sensitivity of 71.2% (stored samples) waslower than a previous report from our laboratory (Kok et al.,1994). In this study the specimens were tested by AgEIAand enhanced culture without prior selection for optimal res-piratory epithelial cell numbers. This may have contributed

to the lower sensitivity of the AgEIA test (Kok et al., 1994;Matthey et al., 1992).

The AgEIA sensitivity (62.5%) in the group of patients(‘fresh’ specimen group) with a median age of 63 years issimilar to the report bySteininger et al., 2002. The latterreport suggested that the lower antigen detection sensitivityof ELISA is a result of decreasing virus concentrations withaccumulating infections and increasing ages. In their report,the ELISA positive rate was 62% (93/150), virus isolation80.6% (121/150) and RT-PCR 93% (80/86) amongst thosewhich were influenza A virus positive in at least one of thethree tests.

Overall, the results of our study with the matrix geneRT-PCR showed the highest sensitivity (100%) in the groupof ‘fresh’ specimens, but this was less sensitive (82.9%)than culture in the group of ‘stored’ specimens. Prompt test-ing on receipt or rapid freezing of samples at−70◦C withthe use of ribonuclease inhibitors may be required to obtainhigh sensitivity with RT-PCR test for RNA viruses in respi-ratory specimens. We have shown that the use of hybridiza-tion probes in the LightCycler real time PCR can provide asensitive detection system for influenza A infection with si-multaneous confirmation of specific amplified sequence andrapid presumptive differentiation of H1N1 and H3N2 in-fluenza A viruses, the predominant circulating strains (Coxand Subbarao, 2000). Rapid and sensitive detection of in-fluenza A virus by RT-PCR, coupled with virus isolation andsubsequent sequence analysis of viral strains, will facilitatediagnosis and strain identification for vaccine production tothis important worldwide infection.

Acknowledgements

We thank the staff of the Virus Detection Laboratory fortheir expert technical assistance and Ms. Kristyn Willson forstatistical analysis.

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Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR

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