Rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool.

We compared the detection of seven respiratory viruses by using a commercially available monoclonal antibody pool in a 2-day shell vial assay with that by using standard cell culture with respiratory syncytial virus (RSV) enzyme-linked immunosorbent assay (ELISA)-negative nasal secretions from hospitalized children. We found 179 respiratory virus isolates by either method in 675 specimens. Overall, the shell vial assay detected 147 of 179 (79%) of the positives after 2 days; cell culture detected 148 of 179 (80%) after a mean incubation period of 7.6 days (range, 1 to 14 days). The sensitivity of the shell vial assay was 78% for RSV, 94% for influenza B virus, 83% for adenovirus, and 80% for parainfluenza viruses. The sensitivity of the cell culture was 70% for RSV, 79% for influenza B virus, 90% for adenovirus, and 89% for parainfluenza viruses. The 2-day shell vial assay allowed the detection of respiratory viruses in a clinically relevant time frame and rapidly detected RSV in specimens lacking RSV antigen by ELISA.     

Diagnosis of respiratory tract viruses in 24 h by immunofluorescent staining of shell vial cultures containing Madin-Darby Canine Kidney (MDCK) cells.

Nine hundred and seventy-eight clinical specimens were examined taken from patients with respiratory tract viruses (RV)-like syndrome between November 1996 and July 1998. The study was undertaken to evaluate the effectiveness of centrifuge-enhanced shell vial cultures (SVC) containing Madin-Darby Canine Kidney (MDCK) cells, combined with immunofluorescent (IF) staining in 24 h. This technique rapidly detects and identifies respiratory tract viruses. The conventional tube culture system with multiple cell lines would ordinarily detect RV within 3-30 days. The SVC/IF method using single cell line (MDCK cells) allowed detection of 81.5% of influenza A virus, 72% of parainfluenza virus, 82.6% of respiratory syncytial virus (RSV) and 79.6% of adenovirus in 24 h.     

Rapid detection of respiratory viruses using mixtures of monoclonal antibodies on shell vial cultures.

Eleven hundred and thirty-three clinical specimens submitted to the laboratory for diagnosis of respiratory virus infections were tested by direct immunofluorescence (DIF) for respiratory syncytial virus (RSV), by shell vial culture, and by conventional cell culture. The shell vial cultures were stained with 8 different monoclonal antibodies both 1 day and 3-7 days after inoculation. In order to limit the cost and the workload, mixtures of monoclonal antibodies were used. Coverslips with HEp-2 cells were incubated with a mixture of FITC-labeled monoclonal antibody to RSV and nonlabeled monoclonal antibody to adenovirus. When no RSV positive IF staining was observed after the first incubation step, the same coverslip was incubated once more with FITC-labeled anti-mouse antibody. A positive reaction at this stage indicated the presence of adenovirus. Similarly, cultures of tertiary monkey kidney cells were investigated with a mixture of two FITC-labeled monoclonals to the influenza viruses A and B and three nonlabeled monoclonals to the parainfluenza viruses 1, 2 and 3. If influenza virus or parainfluenza virus was detected, the exact type was determined by staining different parts of a duplicate coverslip. Shell vial cultures for cytomegalovirus (CMV) were always performed separately on human embryonic lung fibroblasts. Using this approach, we detected RSV (n = 248), CMV (n = 42), parainfluenza virus (n = 31), influenza virus (n = 28), and adenovirus (n = 6), in most cases after only one day of culture. For RSV, the sensitivity of the shell vial method was too low (74%) to allow omission of DIF (sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line

Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.     

Evaluation of the Prodesse Hexaplex multiplex PCR assay for direct detection of seven respiratory viruses in clinical specimens

We evaluated the Hexaplex assay (Prodesse, Waukesha, WI) for the detection of 7 respiratory viruses (influenza A and B, parainfluenza 1-3, and respiratory syncytial virus [RSV] A and B). The Hexaplex assay was performed on 300 respiratory samples during the 1999-2000 respiratory virus season. Results of this assay were compared with shell vial cell culture and/or direct fluorescent antibody stain. The overall sensitivity and specificity of the assay were 96.6% and 94.1%, respectively. The respective sensitivity and specificity of the Hexaplex assay for detection of specific virus groups were as follows: influenza A, 98.6% and 97.8%; influenza B, 100% and 100%; and for parainfluenza viruses (1-3), 100% and 99.1%. The assay did not perform as well with patients infected with RSV: sensitivity and specificity were 91.0% and 98.6%, respectively. There are 2 major drawbacks to this assay: it is technically demanding (3-4 hours hands-on time), and it is expensive ($80-$90 direct cost). Nevertheless, because of the excellent sensitivity and specificity, the Hexaplex assay may be valuable in the diagnosis of respiratory viral infections in immunocompromised patients.     

Rapid and sensitive detection of respiratory virus infections for directed antiviral treatment using R-Mix cultures.

BACKGROUND: The development of new anti-influenza drugs has led to concerns regarding the impact on healthcare costs if they are used indiscriminately. Restricting their use to proven influenza virus infections has the potential to overcome costly inappropriate therapy. However, conventional culture (CC) does not generate results quickly enough to facilitate the timely initiation of treatment, and rapid detection tests have suboptimal sensitivity. We therefore investigated a new rapid culture system (R-Mix) that contains a mixture of two cell lines and detects respiratory viruses within 24 h. OBJECTIVES: To compare the analytical sensitivity of R-Mix with CC and rapid detection methods, for the detection of influenza and other respiratory viruses. To compare the clinical sensitivity of R-Mix with CC and direct antigen detection for the detection of respiratory viruses in primary and acute care settings. STUDY DESIGN: Stock cultures of influenza virus were titrated and tested by R-Mix, ZstatFlu and FLU OIA. Stock cultures of adenovirus and parainfluenza virus type 3 were titrated and tested by R-Mix and CC. Specimens, which had previously tested positive for influenza viruses, were titrated and tested by R-Mix and CC. In symptomatic patients, the majority of whom were from primary care settings, 124 sequential specimens were tested for influenza viruses by immunofluorescent direct antigen detection and R-Mix. A separate set of 111 sequential specimens, from various symptomatic patient groups, were tested for influenza viruses by CC and R-Mix. Additionally, in acute care patients being surveillance tested during periods of immunosuppression, 155 specimens were tested for respiratory viruses (influenza A and B, parainfluenza 1-3, adenovirus and respiratory syncytial virus (RSV)) by CC and R-Mix. RESULTS: With titrated stock cultures, R-Mix showed an analytical limit of detection of ten infectious virus particles per vial for influenza A, compared with 100,000 particles per test for FLU OIA and 1,000,000 for ZstatFlu. R-Mix also showed a 100-fold greater sensitivity for the detection of influenza A and equivalent sensitivity for the detection of influenza B when compared with CC in titrated known positive specimens. Further, it showed equivalent sensitivity to CC for the detection of adenovirus and parainfluenza virus type 3 in titrated stock cultures. Among prospective specimens from symptomatic patients, the sensitivity of R-Mix, CC and direct antigen detection tests (DAT) for influenza virus detection, was 100, 67 and 66%, respectively, and the specificity was 100, 100 and 98%, respectively. In surveillance specimens from immunosuppressed patients, the sensitivities of R-Mix and CC for respiratory virus detection were equivalent. Moreover, R-Mix results were available within 24 h, and by altering the antibody staining reagents either influenza viruses, or all seven major respiratory viruses, could be detected and distinguished in a single test. CONCLUSIONS: R-Mix is a simple, rapid and sensitive system for the detection of influenza viruses that facilitates the restriction of antiviral drugs to patients with culture-confirmed infections.     

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.     

Clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study

To assess the expected benefits of rapid reporting of respiratory viruses, we compared patients whose samples were processed using standard techniques such as enzyme immunoassays, shell vial assays, and culture tube assays (year 1) to patients whose samples were processed with the same standard techniques in addition to immunofluorescent testing (FA) directly on cytocentrifuged samples (year 2). The cytospin FA screened for influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3, and adenovirus (DAKO Diagnostics Ltd.). The specificity of the cytospin FA for all viruses was 100%. The sensitivities for influenza A virus and RSV were 90 and 98%, respectively, but the sensitivities for influenza B virus and adenovirus were unacceptable (14.3 and 0%, respectively). However, since the former viruses account for >85% of our isolates from clinical specimens, the cytospin FA is an excellent screening test since the positive result was available within hours. The mean turnaround time for all positive viruses was 4.5 days in year 1 and 0.9 day in year 2 (P = 0.001). This rapid reporting resulted in physicians having access to information sooner, enabling more appropriate treatment. The mean length of stay in the hospital for inpatients with respiratory viral isolates was 10.6 days for year 1 versus 5.3 days for year 2. Mean variable costs for these patients was $7,893 in year 1 and $2,177 in year 2. After subtracting reagent costs and technological time, the savings in variable costs was $144,332/year. Summarizing, the cytospin FA markedly decreased turnaround time and was associated with decreased mortality, length of stay, and costs and with better antibiotic stewardship.     

Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens

We compared the diagnostic performance and overall respiratory pathogen detection rate of the premarket version of the FilmArray Respiratory Panel (RP) multiplex PCR assay (Idaho Technology, Inc., Salt Lake City, UT) with those of the Food and Drug Administration (FDA)-cleared Prodesse ProFlu+, ProFAST+, ProParaflu+, Pro hMPV+, and ProAdeno+ real-time PCR assays (Gen-Probe, San Diego, CA). The assays were performed on a panel of 192 nasopharyngeal-secretion specimens collected from 81 children under 1 year of age with upper respiratory tract symptoms. To resolve discordant results and confirm pathogens detected only by the larger FilmArray panel, we performed laboratory-developed real-time PCR assays. Among viruses detectable by both commercial assays (adenovirus, human metapneumovirus, influenza A virus, influenza B virus, parainfluenza viruses 1 to 3, and respiratory syncytial virus), the FilmArray and Prodesse assays showed good overall agreement (181/192 [94.3%]; kappa = 0.87; 95% CI, 0.79 to 0.94). FilmArray RP detected more parainfluenza viruses 1 and 3 than ProParaflu+ (18 versus 13) while ProAdeno+ detected more adenoviruses (11 versus 6), but these differences were not statistically significant. Additionally, FilmArray RP detected 138 pathogens (confirmed as true positives) not included in the Prodesse assays (rhinovirus [RV]/enterovirus [EV], 118; bocavirus, 8; coronavirus, 7; parainfluenza virus 4, 4; Mycoplasma pneumoniae, 1). FilmArray RP was cleared by the FDA following the completion of this study. The FDA-cleared version includes the following targets: adenovirus, coronaviruses HKU1 and NL63, human metapneumovirus (hMPV), influenza A virus (to type level only), influenza A H1 seasonal virus, influenza A H3 seasonal virus, influenza A virus H1-2009, influenza B virus, parainfluenza viruses 1 to 4, respiratory syncytial virus (RSV), and RV/EV (no differentiation). The larger panel in the FilmArray RP assay allowed the detection of additional respiratory pathogens compared to the Prodesse assays. In this population of young children with upper respiratory tract infection, RV/EV accounted for the majority of the additional pathogens detected by FilmArray RP.     

SimulFluor respiratory screen for rapid detection of multiple respiratory viruses in clinical specimens by immunofluorescence staining

A new rapid direct immunofluorescence assay (DFA) respiratory screen reagent for detection of seven common respiratory viruses (respiratory syncytial virus [RSV], influenza A and B viruses, parainfluenza virus types 1 to 3, and adenovirus) was compared with standard single or dual DFA reagents and culture. In total, 1,531 respiratory samples were adequate for testing with both SimulFluor Respiratory Screen (RS) reagent (Chemicon International, Temecula, Calif.) and single or dual DFA reagents. The RS DFA reagent detected 367 (98.4%) and single or dual DFA reagents detected 368 (98.7%) of 373 DFA-positive samples. In addition, the RS DFA reagent was equivalent to or better than culture for detection of all viruses except adenovirus. Only 15 of 799 (1.9%) RS-negative samples inoculated into cell cultures yielded respiratory virus isolates (one RSV, five influenza A virus, two influenza B virus, one parainfluenza virus, and six adenovirus). Sixty-six other virus isolates (13 rhinovirus, 24 cytomegalovirus, 28 herpes simplex virus type 1, and 1 enterovirus) were also recovered in culture. With cytospin preparation of slides, only 7.5% of samples submitted were deemed inadequate for DFA. The availability of a rapid DFA screening reagent for detection of multiple common respiratory viruses within 1 to 2 h of sample collection should be of great benefit in terms of patient management and infection control.     

重组人干扰素α-2b喷雾剂预防SARS等呼吸道病毒感染的人群试验研究

目的评估重组人干扰素α2b喷雾剂(远策素喷雾剂)预防SARS等常见呼吸道病毒感染的效果。方法研究对象共14391人,用药剂量为90万IU/次,每日2次,连用5d,未次用药后15d取血,或用药前和用药3周后采取双份血清。采用随机、对照方法检测血清抗SARSCoVIgG抗体;采用双盲、随机、安慰剂对照方法测定血清抗常见呼吸道病毒(B型流感病毒、副流感病毒1～3型,呼吸道合胞病毒及腺病毒3、7型)的血清IgM抗体。结果两次实验中,干扰素组血清SARS病毒IgG抗体阳性率均较试验组高,但差异无统计学意义(P>0.05)。但用药组应用干扰素后副流感病毒1～3型,B型流感病毒,腺病毒3、7型和呼吸道合胞病毒IgM抗体阳性率(依次为6.45%、4.52%、4.30%和17.20%)均低于对照组(依次为19.40%、13.60%、7.12%和25.62%)。其中副流感病毒、B型流感病毒、腺病毒3种病毒IgM抗体阳性率差异均有统计学意义(P<0.01)。结论应用远策素喷雾剂鼻和咽部喷雾能不同程度地降低用药人群常见呼吸道病毒的感染率。     

Simultaneous infections with different enteric and respiratory tract viruses.

Infants and young children with rotavirus (RV) or visualized adenovirus in their stools were tested for the simultaneous presence of a respiratory viral pathogen in their upper respiratory tract. Overall, at least 10.7% of 484 study subjects had such dual infections, including 8.3% of 385 RV-positive gastroenteritis patients and 24.3% of 37 RV-positive respiratory disease patients. Respiratory syncytial virus was present in 34.1% of 41 dual infections with RV and at least 40% of the 12 to 15 dual infections with visualized fecal adenovirus. Other pathogens found in the respiratory tract of patients with RV or visualized fecal adenovirus infections included influenza viruses, adenoviruses, parainfluenza viruses, rhinoviruses, and a cytomegalovirus.     

Enhanced detection of respiratory viruses using cryopreserved R-Mix ReadyCells.

BACKGROUND: R-Mix, which contains a fresh mixture of two cell lines, Mv1Lu (mink lung cells) and A549 cells, has shown good sensitivity and specificity for respiratory virus culture. However, it has until recently only been available in North America, in part due to the shipping constraints associated with cell aging and the difficulty in providing these cells to hard to reach regions. Recently, cryopreserved R-Mix ReadyCells for longer storage were developed. These cells, which are shipped on dry ice and have a shelf life as long as 6 months from date of manufacture, can be thawed and used as needed with minimal addition of refeeding media. OBJECTIVE: Assess the potential for cryopreserved R-Mix ReadyCells to replace conventional culture. STUDY DESIGN: Two hundred and twenty-three nasopharyngeal aspirates confirmed as respiratory virus-positive by conventional culture were inoculated into cryopreserved R-Mix ReadyCells and re-inoculated into conventional culture cells simultaneously. After 1 and 3 days of incubation cryopreserved R-Mix ReadyCells and conventional culture cells were screened using a respiratory virus fluorescent antibody pool for the detection of seven major respiratory viruses (influenza A and B viruses, parainfluenza 1, 2 and 3 viruses, respiratory syncytial virus and adenovirus). Positive pool results were further differentiated with specific monoclonal antibodies against the individual viruses. RESULTS: After 1 day of incubation detection rates for conventional culture were 25%, 39%, 39%, 49%, and 10% for influenza A virus, influenza B virus, parainfluenza viruses, respiratory syncytial virus, and adenovirus, respectively. Corresponding detection rates for cryopreserved R-Mix ReadyCells were 78%, 91%, 72%, 81%, and 65%. Average detection rates of cryopreserved R-Mix ReadyCells for all respiratory viruses were 80% after 1 day incubation and 95% after 3 days incubation, compared to 35% and 70% by conventional culture. CONCLUSION: The cryopreserved R-Mix ReadyCells system offers a highly sensitive and rapid method for detection of respiratory viruses that may allow it to replace conventional cell culture systems.     

Immunofluorescent method of studying autopsy material in acute pneumonia]

The immunofluorescent procedure in examinations of the autopsy material from 304 fatal cases hospitalized for acute pneumonia permitted to diagnose influenza A2 in 29.0%, influenza B in 18.1%, parainfluenza in 3.5%, adenovirus infection in 9.2% and respiratory syncytial virus infection in 3.5% of the cases. In the period of a high incidence of acute respiratory infection, influenza A2 was detected by this method in 40.9% and influenza B in 50% of the cases. Simultaneous examinations of the material in the influenza epidemic period by virological and immunofluorescent methods (63 cases) in 13 cases positive results were obtained with both methods, in 6 cases where influenza viruses were detected the immunofluorescent test was negative, and in 28 cases the positive diagnosis by the immunofluorescent test could not be confirmed virologically. Among the cases examined, 33 were found by the immunofluorescence test to have a mixed respiratory infection, including influenza A2 with other forms of respiratory infection in 18, and influenza B with other respiratory infections in 19 cases. Serological examinations by the complement fixation and hemagglutination inhibition tests on the blood from fatal cases irrespective of the time of examination, as a rule, revealed antibody in low titres which did not confirm the diagnosis.     

Rapid viral diagnosis of acute respiratory infections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions.

Nasopharyngeal secretions from adults and children were obtained in Stockholm, Sweden, for routine diagnosis of influenza A virus, influenza B virus, respiratory syncytial (RS) virus, parainfluenza type 3 virus, and adenovirus infections by demonstration of viral antigens directly in the specimens. The cells in nasopharyngeal secretions were pelleted by centrifugation for preparation of cell deposits for diagnosis by the immunofluorescence technique (IF) in London, England, and in Stockholm, whereas the supernatants were used to diagnose infection by the enzyme-linked immunosorbent assay (ELISA) in Stockholm. Titrations of the various purified viruses showed that ELISA could detect viral antigens in amounts corresponding to 1 to 10 ng of virus protein per test well. In a series of 73 specimens tested for influenza A, RS, and parainfluenza type 3 viruses by IF in London and by ELISA in Stockholm, 15 of 18 RS, 14 of 15 influenza A, and 2 of 2 parainfluenza type 3 viral infections were diagnosed by ELISA as compared with IF, giving sensitivities for RS and influenza A viral diagnosis of 83 and 93%, respectively, and a specificity of 100%. In another series of specimens from 35 patients tested for influenza B virus and adenovirus, five influenza B virus and four adenovirus infections were diagnosed by both methods; one additional influenza B infection was detected only by IF and another only by ELISA. Comparisons of diagnostic results between the two methods performed in Stockholm gave nonagreement of results for 37 of 1,593 tests (2.5%) for the five viruses. The conclusion reached was that the described ELISA, although a satisfactory test, had somewhat less sensitivity than did IF for the detection of respiratory viral infections. This could possibly be explained by unnecessary dilutions of specimens at the time of collection; transportation, processing, and storage of specimens were less complicated than for IF.     

Enhanced detection of respiratory viruses using the shell vial technique and monoclonal antibodies.

The shell vial technique using A549 and MDCK cells, coupled with the use of Bartels respiratory viral monoclonal antibodies, was evaluated initially for the detection of 28 previously isolated respiratory viruses. All viruses were recovered and correctly identified. The shell vial-monoclonal antibody technique was then evaluated for virus isolation from 338 respiratory specimens and compared with the conventional tube method. Both methods gave rise to a total of 83 virus isolates. Of these isolates, 68 (20.1%) were isolated and identified by the shell vial-monoclonal method; 60 (17.8%) were culture-positive by the conventional tube method; forty-five (13.3%) were positive by both methods. The shell vial-monoclonal antibody method yielded 12 isolates of influenza A, two isolates of parainfluenza type 3 and one each of parainfluenza types 1 and 3, which were missed by the conventional tube method, indicating the superior sensitivity and specificity of the shell vial-monoclonal antibody method (Chi-square analysis, P = 0.001) for the detection of these viruses. Of the 50 RSV isolates, 29 were detected by both methods and there were 21 discrepancies between the two methods. The shell vial-monoclonal antibody method also improved the turn-around time for the respiratory virus groups.     

Simultaneous detection of influenza viruses A and B using real-time quantitative PCR

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.