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Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer
Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel
ABSTRACT
The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories.
INTRODUCTION
Influenza viruses are segmented negative-sense RNA viruses that belong to the family Orthomyxoviridae. Three types of influenza viruses, types A, B, and C, have been described on the basis of antigenic differences in the matrix (M) protein and the nucleoprotein. Influenza viruses A and B are associated with seasonal morbidity and mortality, while influenza virus C causes mild upper respiratory tract infections in children and adolescents (17, 18).
Influenza virus A is further classified into different subtypes according to antigenic and genetic differences in its surface glycoproteins. Fifteen hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes have been identified to date. Viruses bearing all known HA and NA subtypes have been isolated from avian hosts; but until recently, only viruses of the H1N1, H2N2, and H3N2 subtypes have been associated with widespread epidemics in humans. During the 2001-2002 influenza season reassortant influenza virus A subtype H1N2 emerged and caused respiratory infections in patients from Israel and other countries (7, 9).
Influenza virus infections are often underdiagnosed and affect patients in all age groups. However, complications of influenza virus infections are often seen in very young individuals, immunocompromised individuals, and elderly individuals. Influenza virus infection has a rapid onset with symptoms that can include fever (temperature, 37.8°C), headache, malaise, cough, chills, myalgia, and sore throat.
Surveillance for influenza viruses in communities is important for providing information concerning the presence of the influenza virus subtype circulating in that community. In addition, with the advent of the new antiviral drugs with activities against influenza viruses A and B (NA inhibitors), successful treatment of influenza virus infections should be initiated within 48 h of the start of symptoms (4). The use of antiviral drugs will decrease the duration of an outbreak, lower costs due to the illness, and prevent the inappropriate use of antibiotics (1, 3). Therefore, a rapid and sensitive means for the detection of both influenza viruses A and B is desired.
Classical diagnosis of influenza virus infections involves viral isolation in tissue culture (TC) on primary monkey kidney cells or Madin-Darby canine kidney (MDCK) cells. However, this method is very tedious and cumbersome, and on average, it takes 3 to 21 days to isolate the virus (11). More rapid antigen detection methodologies have been developed and evaluated. Several immunofluorescence assays, optical immunoassays, and enzyme-linked immunosorbent assays for the direct detection of influenza virus are available commercially (12, 14, 15, 27). However, these assays lack the sensitivity (50 to 80%) required for the rapid detection of these viral pathogens. Moreover, specimen integrity and the presence of sufficient numbers of intact cells in the specimen are crucial for reliable direct immunofluorescence assay results (11).
Rapid and sensitive molecular diagnostic techniques (reverse transcriptase PCR [RT-PCR]) for the detection of influenza viruses have been developed and evaluated (6, 13, 20). However, these assays are technically demanding and prone to contamination, since the reaction tubes are opened after the amplification step. Real-time RT-PCRs (which use TaqMan, LightCycler, and SmartCycler chemistries) for the detection of influenza viruses in clinical samples have recently been described (10, 22, 24, 26). These assays use a fluorescent probe for the simultaneous amplification and detection of the PCR product.
In this study we compared the performances of regular RT-PCR, a shell vial indirect immunofluorescence assay (SV-IFA), and TC with that of the real-time multiplex RT-PCR (with TaqMan chemistry) with patient samples used for surveillance for influenza viruses in Israel. Three hundred seventy clinical samples from patients were analyzed by the four different diagnostic assays during the 2001-2002 influenza season for the presence of influenza virus A or B by using two different fluorescent probes (6-carboxyfluorescein and VIC).
MATERIALS AND METHODS
Epidemiology. The Israel Center for Disease Control, together with the National Center for Influenza in the Central Virology Laboratory and a group of pediatricians and family physicians in primary care services from the central and northern parts of the Israel, established a sentinel system for the reporting of influenza virus activity (19). Surveillance data for morbidity from influenza comprised clinical information from patients with influenza-like illnesses and were backed by laboratory analysis of respiratory specimens.
Clinical specimens. A throat swab specimen and two nasal swab specimens, one from each nostril, were collected from each of 370 individuals presenting with influenza and influenza-like illnesses at sentinel patient clinics throughout Israel. Health care providers were educated on the proper methods of specimen collection, storage, and transport. All three swab specimens collected from each patient were placed in one viral transport tube. The swabs were kept at 4°C and were transported within 48 h to Israel's Central Virology Laboratory in medium 199 supplemented with 200 μg of streptomycin per ml, 100 U of penicillin per ml, and 121.5 U of nystatin per ml at 4°C. On receipt of the specimen, prior to any manipulation of the specimen, an aliquot was removed in a type II biological safety cabinet for molecular analysis and was stored at –70°C until it was tested. Molecular testing was performed either on the same day or the day after receipt of the sample. Patient samples were tested by TC on the day that the specimen was received.
Virus and bacterial stocks. The following reference virus strains were obtained from the American Type Culture Collection (ATCC; Manassas, Va.): influenza virus B/Lee/40 (ATCC VR-101); influenza virus A/Hong Kong/8/68 (H3N2) (ATCC VR-544); adenovirus type 2 (ATCC VR-846), parainfluenza viruses 1 (ATCC VR-94), 2 (ATCC VR-92), and 3 (ATCC VR-93); respiratory syncytial virus (ATCC VR-1302); and coxsackievirus group A24 (ATCC VR-583). In addition, clinical isolates of influenza virus A/140/01 H1N1 and cytomegalovirus-, Epstein-Barr virus-, and human immunodeficiency virus-positive patient samples from the year 2001 were used in the study. Avian influenza virus (H7N7, H5N3, and H9N2) RNAs were kindly provided by Ester Shihmanter from the Israel Veterinary Institute, Beit Dagan. Bacterial isolates from patients, including Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumoniae, and Staphylococcus aureus, were kindly provided by Gil Smollen from the Bacteriology Laboratory at Sheba Medical Center.
Cell line, virus isolation, and identification. MDCK cells (MRC 201129) were generously provided by the World Health Organization Medical Research Council, London, United Kingdom. Cell monolayers in roller tubes were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 200 μg of streptomycin per ml, 100 U of penicillin per ml, and 121.5 U of nystatin per ml in a 37°C incubator. Patient samples were vortexed for 30 s to resuspend the clinical sample trapped on the swab, and 200 μl of the patient sample was used to inoculate MDCK cells in duplicate. Inoculated cells were maintained in DMEM supplemented with 200 μg of streptomycin per ml, 100 U of penicillin per ml, 121.5 U of nystatin per ml, and 2 μg of trypsin type IX (T-0.134; Sigma) per ml and were incubated at 34°C. Cell cultures were observed for cytopathic effects (CPEs) daily for 14 days. Hemagglutination with turkey red blood cells (0.5%; vol/vol) and guinea pig red blood cells (0.75%; vol/vol) was performed with samples suspected of having a CPE. If no CPE was observed, hemagglutination was performed continuously until the end of the incubation period. Every hemagglutination-positive culture was identified by using the classical hemagglutination inhibition procedure (2). Hemagglutination inhibition was performed with influenza virus antisera (A/New Caledonia/20/99, A/Panama/2007/99, B/Hong Kong/22/22001, and B/Victoria/504/2000), which were obtained from the World Health Organization Collaborating Center for Influenza, Centers for Disease Control and Prevention (Atlanta, Ga.).
SV-IFA. MDCK cells in 96-well plates were inoculated with the patient samples, incubated, and stained for influenza viruses A and B. Briefly, 100 μl of 2.5 x 105 cells/ml suspended in DMEM with heat-inactivated 2% fetal bovine serum was inoculated into different wells and incubated at 34°C for 4 to 5 h. After the cells settled, 50 μl of DMEM containing 2 μg of trypsin ml was added. Each patient sample (50 μl) was then inoculated into two wells on the same plate. The plates were then centrifuged at 700 x g for 1 h at room temperature and incubated at 34°C for 48 h. The cells were then fixed with 80% ice-cold acetone in phosphate-buffered saline. Influenza virus A and influenza virus B antigens were detected with primary reference mouse monoclonal antibodies for influenza viruses A and B supplied by the Centers for Disease Control and Prevention and with secondary antibodies consisting of a fluorescein isothiocyanate-conjugated F(ab)2 fragment of rabbit anti-mouse immunoglobulin (Dako-Denmark). A Zeiss inverted fluorescent microscope was used to examine the samples for positively staining cells.
RNA extraction. Viral genomic RNA was extracted from the supernatants of the patient samples by using a QIAamp RNA extraction kit (Qiagen GmbH, Hilden, Germany), according to the protocol suggested by the manufacturer. Briefly, clinical samples were homogenized by vortexing for 30 s, and 140 μl was used for the extraction of viral genomic RNA. The RNA was eluted from the columns with 50 μl of elution buffer. The RNA was immediately stored at –70°C after a 5-μl aliquot was used for the one step RT-PCR or TaqMan reaction. Bacterial RNA was extracted with an RNeasy Mini kit (Qiagen).
Multiplex RT-PCR with gel detection. The primers used in this study were previously reported by van Elden et al. (26). Influenza virus A-specific primers were selected to amplify part of the influenza virus M-protein gene (nucleotide positions 217 to 405). The sequences of the influenza virus A-specific forward and reverse primers are as follows: primer INFA-1, 5'-GGA CTG CAG CGT AGA CGC TT-3'; primer INFA-2/3, 5'-CAT yCT GTT GTA TAT GAG GCC CAT-3'. On the other hand, influenza virus B-specific primers amplified part of the HA gene (nucleotide positions 970 to 1139). The sequences of the influenza virus B-specific forward and reverse primers are as follows: primer INFB-1, 5'-AAATAC GGT GGA TTA AAT AAA AGC AA-3'; primer INFB-2, 5'-CCA GCA ATA GCT CCG AAG AAA-3'. Multiplex RT-PCR was performed by using a One-Step RT-PCR kit (Qiagen). Briefly, 5 μl of extracted RNA was added to a master mixture composed of an enzyme mixture (heterodimeric recombinant RTs Omniscript and Sensiscript and HotStart Taq DNA polymerase), 400 μM each deoxynucleoside triphosphate, 20 U of RNase inhibitor (CPG Inc., Lincoln Park, N.J.), and a mixture of influenza virus A-specific and influenza virus B-specific primers, each at a final concentration of 20 pmol. The optimized profile in the thermal cycler (PTC-100; MJ Research Watertown, Mass.) was 50°C for 30 min and 95°C for 15 min, followed by 35 amplification cycles (with each cycle consisting of denaturation at 94°C for 45 s, annealing at 56°C for 45 s, and synthesis at 72°C for 1 min). Amplification was completed with a prolonged synthesis at 72°C for 10 min. Amplicons were visualized by ethidium bromide staining following electrophoresis on a 2% agarose gel. Analysis of discrepant results was performed by using the primers described by Poddar (20), in which the influenza virus A-specific primers were selected to amplify part of the influenza virus M-protein gene (nucleotide positions 65 to 400), while the influenza virus B-specific primers were selected to amplify part of the NS gene (nucleotide positions 37 to 145).
TaqMan RT-PCR. An ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, Calif.) was used for amplification and detection of influenza virus A and B amplicons. The primers and probes specific for influenza viruses A and B used in this study were previously described by van Elden et al. (26). In our study the influenza virus A-specific probe was labeled with FAM at the 5' end (FAM-5'-CTC AGT TAT TCT GCT GGT GCA CTT GCC A-3'-TAMRA, where TAMRA is the quencher dye 6-carboxytetramethylrhodamine), while the influenza virus B-specific probe was labeled with VIC at the 5'end (VIC-5'-CAC CCA TAT TGG GCA ATT TCC TAT GGC-3'-TAMRA). VIC is different from the dye used by van Elden et al. (26). Both probes had the quencher dye TAMRA at their 3' ends. The sensitivity of the TaqMan assay was optimized by evaluating different concentrations of primers (200, 300, 600, and 900 nM) and probes (100, 200, and 300 nM). The concentrations of primers used in this study that gave the best detection limits were 300 nM for influenza virus A-specific primers, 600 nM for influenza virus B-specific primers, and 200 nM for the influenza virus A-specific (FAM) and influenza virus B-specific (VIC) probes. The real time RT-PCR conditions used in this study were similar to those used by van Elden et al. (26), with some modifications. Unlike the two-step RT-PCR reported by van Elden et al. (26), we used a one-step RT-PCR procedure to minimize the chance of contamination. Briefly, the 50-μl RT-PCR mixture contained 25 μl of 2x TaqMan One-Step RT-PCR master mixture reagents (Applied Biosystems) containing 5-carboxy-X-rhodamine, succinimidyl ester (ROX) as an internal reference dye, 1.25 μl of a MultiScribe and RNase inhibitor mixture, 300 nM each influenza virus A-specific primer, 600 nM each influenza virus B-specific primer, and 200 nM each probe. Amplification and detection were performed under the following conditions: 30 min at 48°C for reverse transcription, 10 min at 95°C to activate AmpliTaq Gold DNA polymerase, and 50 cycles of 15 s at 95°C and 1 min at 60°C.
During amplification the ABI 7700 Prism sequence detector monitored the fluorescence emission once every 7 s. ROX was used as a passive reference to whose signal the signal of the reporter dye was normalized during data analysis, which reduced the non-PCR-related fluorescence fluctuation from well to well. The cycle threshold (CT) represents the cycle number at which significantly increased fluorescence is first detected. Analysis of discrepant results was performed as described above in the section on multiplex RT-PCR with gel detection.
RESULTS
Detection limits. The detection limits for influenza viruses by each of the assays, regular RT-PCR, RT-PCR with TaqMan chemistry, SV-IFA, and TC, were determined with a series of serial dilutions in DMEM of influenza viruses A/Hong Kong/8/68 (H3N2) (ATCC VR-544) and B/Lee/40 (ATCC VR-101) with known 50% chick embryo infectious doses (CEID50s). Aliquots from each dilution were taken for infectivity testing with MDCK cells in roller tubes and SV-IFAs with MDCK cells. In addition, nucleic acids were extracted from an aliquot of each sample on the same day for molecular testing. The detection limits of all four assays were evaluated three times, with identical results each time. The results are summarized in Table 1.
Multiplex one-step RT-PCR with gel detection amplified products of influenza viruses A and B at 3.9 and 4.4 CEID50s, respectively. There was no difference in the detection limit of the assay when the primers were used in a single or a multiplex assay.
The TaqMan assay was more sensitive for the detection of influenza virus A (0.039 CEID50s) than RT-PCR with gel detection. In contrast, the detection limits of the TaqMan assay and RT-PCR with gel detection for influenza virus B were identical (4.4 CEID50s), whether RT-PCR was performed as a single or a multiplex TaqMan assay. The multiplex assay was capable of immediate distinction between influenza A virus and influenza B virus due to the different fluorophors attached to the probes (FAM for influenza A virus and VIC for influenza B virus).
The detection limit of TC with MDCK cells for influenza virus A was comparable to that of the one-step RT-PCR with gel detection, but TC with MDCK cells was 2 log units less sensitive than the TaqMan assay. TC was 2 log units less sensitive than the two molecular biology-based assays for the detection of influenza virus B.
SV-IFA detected 1.4 x 103 CEID50s of influenza virus A and 1.6 x 105 CEID50s of influenza virus B. SV-IFA was 103 to 105 times less sensitive than the other assays evaluated.
The results presented above indicate that the detection limit of the regular one-step RT-PCR was similar to that of TC for influenza virus A. In contrast, RT-PCR was 2 log units more sensitive than TC for the detection of influenza virus B. The TaqMan assay was the most sensitive assay, since the detection limit for influenza virus A was 2 log units better than that of the regular RT-PCR, while the detection limit of TC was similar to that of the regular RT-PCR for influenza virus B.
Cross-reactivity with other respiratory pathogens. Each of the molecular biology-based assays was evaluated for possible cross-reactivity with a number of DNA and RNA viruses. Genetic material from clinical isolates of rhinovirus, enterovirus, cytomegalovirus, human immunodeficiency virus, and Epstein-Barr virus and ATCC isolates of coxsackievirus group A24, adenovirus type 3, parainfluenza viruses types 1 to 3, and respiratory syncytial virus were evaluated. None of these viruses cross-reacted in either the regular RT-PCR or the TaqMan assays. In addition, genetic material of bacterial clinical isolates K. pneumoniae, H. influenza, S. pneumoniae, S. pyogenes, S. aureus, and Escherichia coli did not cross amplify with the influenza virus-specific primers.
Influenza virus strains isolated during the study period. During the 2001-2002 influenza season, 52% of patient samples submitted for influenza virus surveillance were positive by TC. Of these, 63.9% were positive for influenza virus A and 36.1% were positive for influenza virus B. Five strains of influenza viruses were identified by hemagglutination inhibition assays at the Israel Central Virology Laboratory: 93 isolates of an A/Moscow/10/99 (H3N2)-like virus, 28 isolates of an A/Israel/152/01 (H1N2)-like virus, 1 isolate of an A/New Caledonia/20/99 (H1N1)-like virus, 65 isolates of B/Sichuan/379/99-like virus, and 4 isolates of a B/Victoria/2/87-like virus. The main activities of A/Moscow/10/99 (H3N2)-like and A/Israel/152/01 (H1N2)-like influenza viruses were detected between week 51 of 2001 and week 8 of 2002. Influenza virus B/Sichuan/379/99-like activity occurred sporadically throughout the season, while influenza virus B/Victoria/2/87-like activity occurred between weeks 7 and 17 of 2002. The one A/New Caledonia/20/99 (H1N1)-like virus was detected during the month of December 2001. All different subtypes of influenza viruses circulating in Israel were detected by the regular RT-PCR and the TaqMan assays.
It is noteworthy that van Elden et al. (26) have shown that the TaqMan assay detects a wide variety of influenza virus A and B subtypes. We evaluated the capability of the multiplex assay to detect several influenza A virus subtypes that were isolated in previous or more recent years (A/Sydney/99/00 [H3N2], A/Panama/2007/99 [H3N2], an A/Fujian/411/2002 [H3N2]-like virus, A/Wyoming/3/2003 [H3N2], B/Yamanashi/166/98 [a B/Bejing/184/93-like virus], B/Hong Kong/330/01, B/Shandong/7/97, B/Brisbane/32/02, and B/Shanghai/361/02). Various influenza virus subtypes that circulated in Israel between 2000 and 2004 were detected by the TaqMan assay. These included five H3N2 subtypes, seven influenza B subtypes, one H1N1 subtype, and one H1N2 subtype.
Comparison of the multiplex real-time RT-PCR (TaqMan assay) to TC with MDCK cells as a diagnostic assay. Three hundred seventy patient samples were collected during the 2001-2002 influenza season. Overall, 214 patient samples were positive for influenza viruses by the multiplex TaqMan assay, while 191 patient samples were positive by TC. Three patient samples were negative by the TaqMan assay but positive by TC. The total sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 98.4, 85.5, 87.9, and 98.1%, respectively (Table 2).
Upon analysis of the performance of the multiplex TaqMan assay for the detection of influenza viruses A and B, 144 samples were positive for influenza virus A and 70 samples were positive for influenza virus B. One hundred twenty-two samples were positive for influenza virus A and 66 positive were for influenza virus B by TC. Three samples were negative for influenza virus B by the TaqMan assay but positive by TC. The sensitivity, specificity, PPV, and NPV of the TaqMan assay for the detection of influenza virus A were 100, 91.1, 84.7, and 100%, respectively, while those of the TaqMan assay for the detection of influenza virus B were 95.7, 98.7, 94.3, and 99%, respectively (Table 2).
Resolution of discrepant results obtained by TaqMan assay. Seventeen of the 22 samples that were positive for influenza virus A by the TaqMan assay but negative by culture were also positive by regular RT-PCR with the same primer sets used in the TaqMan assay. Upon retesting of the 22 RNA samples with a different set of primers derived from the M-protein gene (20), all 22 samples were positive. Moreover, the original 22 samples positive for influenza virus A but negative by TC were reextracted and tested again by the TaqMan assay and RT-PCR with the two sets of primers whose sequences were derived from the sequence of the M-protein gene. Seventeen samples were positive for influenza virus A by the standard RT-PCR, and the other five were positive with the second set of the M-protein gene-specific primers, thus confirming the results of the first run and supporting the sensitivity studies that the TaqMan assay is more sensitive than culture.
Four samples positive for influenza virus B by the TaqMan assay but negative by TC were all positive upon retesting by the TaqMan assay and by RT-PCR with a different set of primers whose sequences were based on the sequence of the NS gene (20). In addition, all four samples were positive upon reextraction of the RNA from the original samples and testing of those samples by the TaqMan assay and the standard RT-PCR, in addition to assays with the primer sets based on the NS gene.
Evaluation of three samples that were negative for influenza virus B by the TaqMan assay but positive by TC reveled that the multiplex TaqMan assay failed to detect the virus in these three samples. Once the assay was changed from a multiplex format to a singleplex format, the TaqMan assay was positive for all three samples. This indicates that there was no genetic alteration in the primer or probe annealing regions. All three samples were positive by the standard one-step RT-PCR, confirming that these samples were positive for influenza virus B.
Comparison of standard one-step multiplex RT-PCR to TC with MDCK cells. The standard one-step RT-PCR was evaluated by using the same RNA extracted from the 370 patient samples used to evaluate the TaqMan assay. Of the 370 patient samples evaluated, 213 samples were positive by RT-PCR, while 191 samples were positive by TC. Twenty-two samples were positive by the standard RT-PCR and negative by TC. The total sensitivity, specificity, PPV, and NPV were 100, 87.7, 89.7, and 100%, respectively (Table 2). Upon evaluation of the performance of the multiplex one-step RT-PCR assays for the detection and differentiation of influenza viruses A and B, 139 patient samples were positive for influenza virus A, while only 122 were positive by TC. The sensitivity, specificity, PPV, and NPV of the multiplex one-step RT-PCR for the detection of influenza virus A were 100, 93.1, 87.8, and 100%, respectively (Table 2).
The one-step RT-PCR was as sensitive for the detection of influenza virus B as it was for the detection of influenza virus A. Seventy-four patient samples were positive for influenza virus B by the multiplex RT-PCR, while only 69 patient samples were positive by TC. Five patient samples were positive by the multiplex RT-PCR but negative by TC. The sensitivity, specificity, PPV, and NPV of the multiplex one-step RT-PCR for the detection of influenza virus B were 100, 98.3, 93.2, and 100%, respectively (Table 2). Resolution of the discrepant results was identical to that for the TaqMan assay analysis, thus confirming that the RT-PCR results were true positive.
Comparison of SV-IFA to TC with MDCK cells. The overall performance of SV-IFA for the detection of influenza viruses A and B was inferior to those of all other methods. The 370 patient samples evaluated in this study were tested for influenza viruses by SV-IFA. Of the 188 patient samples confirmed to be positive for influenza viruses by TC, 126 samples were positive by SV-IFA, while 65 samples were negative. Three samples were positive by SV-IFA and negative by TC. The total sensitivity, specificity, PPV, and NPV of SV-IFA for the detection of influenza viruses were 65.9, 98.3, 97.7, and 73%, respectively (Table 2).
Upon evaluation of the sensitivity of SV-IFA for the detection of each of the influenza viruses A and B, the assay detected influenza virus A in 103 of 122 patient samples. The sensitivity, specificity, PPV, and NPV of SV-IFA for the detection of influenza virus A were 84.4, 98.8, 97.2, and 92.8%, respectively (Table 2). Evaluation of SV-IFA for the detection of influenza virus B reveled that the assay's performance was very poor. Of the 69 patient samples positive by TC, 23 were positive by SV-IFA and 46 were negative. The sensitivity, specificity, PPV, and NPV of SV-IFA for the detection of influenza virus B were 33.3, 100, 100, and 86.7%, respectively (Table 2).
Comparison of standard RT-PCR to multiplex TaqMan assay. The overall sensitivity and specificity of the one-step RT-PCR assay were compared to those of the TaqMan assay for the detection of influenza viruses A and B from clinical specimens. Of the 370 patient samples analyzed, 209 samples were positive for either of the influenza viruses by both methods and 5 samples were positive by the TaqMan assay but negative by RT-PCR, while 4 samples were positive by RT-PCR but negative by the TaqMan assay. The total sensitivity, specificity, PPV, and NPV of the standard RT-PCR for the detection of influenza viruses compared to the results of the TaqMan assay were 97.7, 97.4, 98.1, and 96.8%, respectively (Table 3). One patient sample that was positive for influenza virus B by standard RT-PCR and negative by the TaqMan assay was also positive by assay with the NS gene primers of Poddar (20). These results allowed us to use the regular one-step RT-PCR as a backup for the TaqMan assay.
Results for standardized quality control samples by the influenza virus TaqMan assay. The influenza virus-positive controls used with each TaqMan assay run were standardized in our laboratory. The positive and the negative controls were extracted side by side, with a maximum of 12 clinical samples used in each extraction run. By minimizing the number of samples extracted simultaneously, we limited the chance of cross contamination, since a large number of the surveillance samples had high concentrations of influenza viruses. The result of the TaqMan assay for the extraction-positive control was monitored, and any deviation of the CT by more than 1 standard deviation (SD) from the established mean CT value for that control lot number resulted in the repetition of the extraction and the TaqMan run. The value of 1 SD was chosen due to the stability of the extraction controls, which are prepared in-house. Over a 6-month period and after 47 consecutive TaqMan runs, the mean CT for the influenza virus A (H1N1) control was 33.3, while the SD and the coefficient of variation (CV) were 0.9 and 2.7%, respectively. For influenza virus A (H3N2), the mean CT was 30.2, while the SD and CV were 0.8 and 2.8%, respectively. On the other hand, for influenza virus B, the mean CT was 34.7 and the SD and the CV were 0.8 and 2.2%, respectively.
The amplification-positive controls (RNAs of influenza virus A H3N2 and H1N1 and influenza virus B) were divided into aliquots and kept frozen at –70°C. Each control was thawed and used only once. The results for these three controls were used to validate each run, and any deviation of more that 1 SD from the mean resulted in repetition of the assay. These controls, which were used as internal quality controls for the TaqMan assay, were highly stable; and the results for these controls very rarely deviated from the mean reading by more than 1 SD. Over the 47 consecutive runs, only one run was repeated since two of the three control CTs were higher than 1 SD from the mean CT.
DISCUSSION
Rapid detection of influenza viruses is becoming of the utmost importance, especially with the advent of antiviral drugs that are potent and specific for influenza viruses A and B and the emergence of highly pathogenic viruses. Infection with these highly pathogenic viruses can result in symptoms that mimic those of the common circulating influenza viruses (H3N2 and H1N1). The emergence of the severe acute respiratory syndrome coronavirus and highly pathogenic avian influenza virus H5N1 further emphasize the importance of the availability of a rapid and sensitive diagnostic assay for the detection of influenza viruses.
Several RT-PCR methods for the detection of influenza viruses A and B have been described. These methods rely either on the analysis of the PCR products size by gel electrophoresis or on the detection of the PCR product by 96-well plate hybridization (5, 6, 13). Agarose gel detection and plate hybridization are labor-intensive and time-consuming, in addition to being prone to cross contamination. Newer approaches that use specific oligonucleotide hybridization on microchips, fluorescence resonance energy transfer probes on LightCycler instruments, and the 5' nuclease PCR or TaqMan chemistry have been described (16, 23, 24, 26). These assays allow the simultaneous amplification and detection of influenza virus nucleic acid in real time.
In this study we evaluated a real-time multiplex assay (which uses the TaqMan chemistry) for rapid surveillance for influenza viruses A and B during the 2001-2002 influenza season in Israel. The study evaluated the performances of previously published primers (26); however, a few modifications to the assay described in the original report (26) were made in order to increase the sensitivity of the assay and to be able to differentiate influenza viruses A and B. Briefly, unlike the original report, in which the cDNA was prepared first and then the PCR was performed, the assay described in this report is a one-step RT-PCR assay. Furthermore, the influenza virus B-specific probe was labeled with VIC and not FAM, as in the study by van Elden et al. (26), in order to be able to differentiate the two viruses.
Unlike the original report by van Elden et al. (26), in which the sensitivities of the assay for influenza viruses A and B were 11 and 13 virus copies, respectively, we observed a 2-log-unit difference in the limits of detection of influenza viruses A and B. This 2-log-unit difference could be due to the optimization of our assay for the detection of very low CEID50s. Moreover, the differences in sensitivity could be because van Elden et al. (26) determined the detection limit of the assay with sucrose gradient-purified virus, while we used reference strains from ATCC with known CEID50s. Others have previously reported differences in the sensitivities of multiplex assays. Templeton et al. (25) reported that the detection limit of their multiplex assay with the iCycler IQ real-time detection system was 2 log units more sensitive for the detection of influenza virus B than of influenza virus A.
The sensitivity and specificity of the TaqMan assay were compared to those of the regular one-step RT-PCR with gel detection with the same primer sets, SV-IFA, and virus isolation by TC with MDCK cells. Evaluation of the sensitivity of the multiplex TaqMan assay showed that it detected a larger proportion of influenza virus A isolates than regular RT-PCR, TC, and SV-IFA did, consistent with other reports that showed that real-time RT-PCR in general has a higher sensitivity than other techniques (20, 22, 16, 26). The increased sensitivity of the TaqMan assay was evident, as the assay detected 22 more positive samples than TC and 5 more positive samples than regular RT-PCR. The 22 samples were also positive by the regular RT-PCR with the primer sets of either van Elden et al. (26) or Poddar (20). The five samples that were positive by the TaqMan assay and negative by RT-PCR were positive with the primer sets of Poddar (20). In addition, the clinical samples came from patients who had clinical symptoms of influenza virus infection, further confirming that the samples were true positives. The lower number of isolates detected by TC could be due either to the low number of infectious viral particles in the sample or to virus inactivation by improper specimen collection, storage, and transport. Improper specimen collection, storage, and transport are highly unlikely, however, since the health care providers were instructed on how to collect, store, and transport the patient samples.
The multiplex real-time TaqMan assay for influenza virus B was not as good (sensitivity, 95.7%; specificity, 98.7%). The multiplex TaqMan assay missed four influenza virus B-positive samples that were positive by culture and regular RT-PCR, despite the comparatively low detection limit. These four samples were true positives and did not have a genetic alteration at the annealing site of the probe, since a change of the multiplex condition to the singleplex condition allowed the detection of virus in these samples. Several reports have described singleplex assays as being more sensitive than multiplex assays, especially with patient samples with low copy numbers. Grondahl et al. (8) suggested that this observation could be due to the accumulation of by-products through nonspecific annealing during PCR if the annealing temperature is suboptimal for some of the templates. In addition, Templeton et al. (25) have reported that multiplex assays for parainfluenza virus 4 and other respiratory pathogens reduces the sensitivity of the multiplex assay by 1 log unit. The virus concentrations in these four samples were low, as evident by the RT-PCR product band on the gel. Smith et al. (24) reported that the sensitivity of influenza virus B detection by use of the LightCycler methodology was similar to that of TC. However, they noted that the number of samples that they used was very small and that a larger number of clinical samples should be tested to better evaluate the performance of the assays. Moreover, performance of the assay as a one-step RT-PCR and not as a two-step RT-PCR might have played a role in the reduced sensitivity of the TaqMan assay for the detection of influenza virus B.
The overall specificity of the TaqMan assay was calculated on the basis of the total number of samples negative for both influenza viruses A and B. The specificity for each influenza virus type was calculated on the basis of the total number of samples negative for the virus for which tests were being conducted.
The overall sensitivity of SV-IFA for the detection of influenza viruses was poor (65.9%). The sensitivities of SV-IFA for the detection of influenza viruses A and B were 84.4 and 33.3%, respectively. This is consistent with the high detection limit (1,400 and 158,000 CEID50s for influenza viruses A and B, respectively). The poor sensitivity of SV-IFA was consistent with the results of many rapid influenza virus antigen detection assays reported by other laboratories (14, 21). The best patient samples for use in the detection of viral respiratory pathogens are nasopharyngeal aspirates. The low sensitivity of SV-IFA could be attributed in part to the fact that the patient samples were a mixture of two nasopharyngeal swab specimens and one throat swab specimen and to the fact that they contained insufficient amounts of infectious virus. Three samples that were positive by SV-IFA but negative by TC were positive by the standard RT-PCR and the TaqMan assays. This suggests that the virus in these samples was inactivated. This could be due to poor sample transport.
The possible cross-reactivity of the TaqMan assay was evaluated with multiple samples of RNA and DNA from different viruses and bacteria. No cross amplification was observed when noninfluenza viruses were tested. Of interest was evaluation of the TaqMan assay detection of avian influenza viruses (H5N3, H7N7, and H9N2) (data not shown). The TaqMan assay gave a positive signal for two avian influenza viruses, H7N7 and H9N2. However, H5N3 was not detected by the TaqMan assay. Moreover, a cDNA that encodes H5N1 of the presently circulating avian influenza virus was not detected by the TaqMan assay. Upon evaluation of the H5N1 RT-PCR product from the TaqMan reaction tube on a 2% agarose gel, a PCR product of the correct size was amplified (data not shown). This indicates that the probe used in the TaqMan assay did not anneal to the H5 PCR product. This is of concern, since this TaqMan assay cannot be used in areas were H5N1 avian influenza virus is circulating. A separate assay for the detection of the avian influenza virus H5N1 is being evaluated in the laboratory.
The sensitivity and specificity of the regular RT-PCR for the detection of influenza viruses A and B were comparable to those of the TaqMan assay. The overall sensitivity and specificity of the regular RT-PCR compared to the results of TC by the use of the Qiagen one-step RT-PCR chemistry were 100 and 87.7%, respectively. The sensitivity and specificity of the regular RT-PCR for the detection of influenza virus A were 100 and 93.1%, respectively, whereas the sensitivity and specificity for the detection of influenza virus B were 100 and 98.3%, respectively. On the basis of these results, the Qiagen one-step RT-PCR is being used as a backup assay for the TaqMan assay when the TaqMan assay machine is down. Once the real-time PCR system was established in the laboratory and the maintenance procedures were followed, the down time was minimal. From our experience with several viral pathogens, a TaqMan assay run can fail, but the machine rarely stops working.
The standardized positive controls developed for use with the TaqMan assay allowed determination of the criteria that could be used for the validation of each run and were also used as internal controls for quality assessment between runs. The homemade controls for the TaqMan assay were very stable at –70°C. This was evident by the performance of the assay daily for several weeks.
Of utmost importance in the performance of any rapid diagnostic molecular biology-based assay is the cost of the diagnostic assay, in addition to the availability of the supporting instrumentation for advanced molecular biology-based diagnosis and well-trained medical technologists. In Israel the Central Virology Laboratory reports the results to the physician in less than 24 h from the time of receipt of the specimen. The estimated direct cost of performing the multiplex influenza virus TaqMan assay in Israel is about $10. Multiplexing of the TaqMan assay reduces the cost of the analysis, whether it is used for clinical diagnosis or for surveillance. Our assay is based on the assay published by van Elden et al. (26), which we have further evaluated using a large number of clinical samples. In addition, our modification, in which we used VIC to label the influenza virus B-specific probe, allowed the immediate discrimination of influenza viruses A and B, reducing the cost even further. However, the lower sensitivity of influenza virus B detection by the multiplex assay needs investigation and improvement.
Moreover, the ability to perform the assay and report the result in less than 24 h from the time of sample receipt enables patients to be treated with antiviral drugs. In addition, the rapid detection of influenza viruses will also help hospitals in their fight against nosocomial infections, since the result can be reported to the physician within 24 h.
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