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.     

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.     

Sensitivity of respiratory virus culture when screening with R-mix fresh cells

Use of R-Mix Fresh Cells has been shown to be a rapid and sensitive method for the detection and identification of respiratory viruses. We prospectively evaluated the impact of incorporation of R-Mix shell vials on the sensitivity and time to detection of seven respiratory viruses recovered in a comprehensive culture during the course of an entire respiratory season in a high-volume clinical laboratory. In this study, R-Mix shell vials were used as part of the culture of 3803 respiratory specimens. A total of 428 respiratory viruses were recovered. Staining of R-Mix vials after overnight incubation allowed initial detection of 274 of 279 influenza viruses, 33 of 38 parainfluenza viruses, 35 of 51 adenoviruses, and 52 of 60 respiratory syncytial viruses (RSVs). The time to reporting of all positive cultures after in-lab specimen receipt was 2.9 days on average and those initially detected in R-Mix cells were reported in 2.3 days on average. A combination of direct fluorescent-antibody (DFA) staining and virus culture was performed on a subset of 711 respiratory specimens. Of 152 viruses identified, 57 were observed only with DFA testing (55 RSV and 2 influenza A viruses) and 31 were recovered only in cell culture. After overnight incubation, R-Mix cells detected 87.1% of respiratory viruses not observed by DFA testing and 96.9% of viruses positive by both methods. The sensitivities of DFA testing and R-Mix cells for identification of influenza viruses were 70.5% and 96.7%, respectively. The R-Mix method detected influenza virus in 18 samples that were negative by DFA testing.     

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 three techniques for detection of respiratory viruses in nasopharyngeal aspirates from children with lower acute respiratory infections

A comparison of immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and isolation in tissue culture (TC) for detection of respiratory viruses was performed on 496 nasopharyngeal aspirates from children under 5 years of age with lower acute respiratory infections who were receiving attention at three hospitals in Buenos Aires, Argentina. All samples were tested by the three methods for respiratory syncytial virus (RSV), influenza A and B, adenovirus, and parainfluenza 1 and 3. Viral diagnosis was made in 167 samples (33.7%); of these, 124 (74.3%) were isolated in TC, whereas 120 (71.8%) were detected by ELISA and 127 (76%) by IF. RSV was detected in 121 samples, mainly by ELISA and IF. The sensitivity and specificity of each rapid technique as compared with isolation in TC were similar, reaching 98% and 92%, respectively. When ELISA was compared with IF, the sensitivity was 95%, and the specificity was 98%. Adenovirus was detected in 18 patients by TC. For this virus, rapid techniques sensitivity as compared with TC was low (almost 22%). Parainfluenza 3 was readily detected by IF and TC; influenza A, B and parainfluenza 1 were detected in few samples; and tissue culture proved more efficient than rapid techniques. The results indicate that both rapid techniques are good tools for the detection of most respiratory viruses except for adenovirus, for which TC cannot be omitted.     

Routine diagnosis of seven respiratory viruses and Mycoplasma pneumoniae by enzyme immunoassay

A composite EIA, using 8-well microstrips, was used for the rapid detection of seven respiratory viruses and M. pnueumoniae. The viruses included influenza A and B, parainfluenza 1, 2 and 3, adenovirus and respiratory syncytial virus. During the 61 month period — June 1988 to June 1993 — 17326 respiratory specimens, submitted from three states, were tested by this EIA. The specimens were mainly from a paediatric population (hospitals and private physicians). RSV was the predominant virus detected, followed by adenovirus, parainfluenza 3, M. pneumoniae, influenza A, parainfluenza 2, influenza B and parainfluenza 1. The use of blocking antibodies confirmed the identification of the agents, in particular with samples showing absorbance values greater than the cutoff with more than one infectious agent. Different methods for processing specimens in order to obtain a uniform suspension, and interpretation of non-specific reactions, are discussed. The assays showed an average sensitivity of 85% and specificity of 99%, compared to virus culture. This EIA system provided an efficient method for the rapid diagnosis of viral and mycoplasmal infections in a busy diagnostic laboratory.     

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.     

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)     

Upper respiratory virus detection without parent-reported illness in children is virus-specific

BACKGROUND: Viral upper respiratory tract infection (vURI) may or may not present with a cold/flu-like illness (CFLI). OBJECTIVES: For common upper respiratory viruses that cause vURIs, to determine the relative frequencies of virus detection by PCR in subjects with and without CFLIs. STUDY DESIGN: Prospective follow-up of 170 children aged 1-8.6 years through the CFLI season by daily parental diary for CFLI episodes and nasal secretion sampling using PCR assays for adenovirus, coronavirus (types 229E and OC43), influenza virus (types A and B), parainfluenza (types 1-3) virus, rhinovirus, and respiratory syncytial virus (RSV). RESULTS: Virus was detected in 415 of 956 independent assays: 425 CFLI episodes and 531 non-CFLI periods were sampled; samples from 270 (64%) CFLI episodes and 145 (27%) non-CFLI periods contained virus detected by PCR. Rhinovirus was most frequently detected at 64%, followed by mixed viruses at 12%, RSV at 7%, and the other viruses at 3-5% of all detections. About 85% of RSV, influenza A and adenovirus detections were associated with a CFLI, whereas less than 62% of other virus detections were associated with CFLI. CONCLUSIONS: The frequency of PCR virus detection without CFLI was different among viruses. This introduces virus-specific biases to estimating the frequencies of specific complications attributable to a vURI when ascertained by CFLI identification.     

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.     

Comprehensive real-time epidemiological data from respiratory infections in Finland between 2010 and 2014 obtained from an automated and multianalyte mariPOC® respiratory pathogen test

Respiratory viruses cause seasonal epidemics every year. Several respiratory pathogens are circulating simultaneously and typical symptoms of different respiratory infections are alike, meaning it is challenging to identify and diagnose different respiratory pathogens based on symptoms alone. mariPOC® is an automated, multianalyte antigen test which allows the rapid detection of nine respiratory infection pathogens [influenza A and B viruses, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, parainfluenza 1–3 viruses and pneumococci] from a single nasopharyngeal swab or aspirate samples, and, in addition, can be linked to laboratory information systems. During the study period from November 2010 to June 2014, a total of 22,485 multianalyte respi tests were performed in the 14 participating laboratories in Finland and, in total, 6897 positive analyte results were recorded. Of the tested samples, 25 % were positive for one respiratory pathogen, with RSV (9.8 %) and influenza A virus (7.2 %) being the most common findings, and 0.65 % of the samples were multivirus-positive. Only small geographical variations in seasonal epidemics occurred. Our results show that the mariPOC® multianalyte respi test allows simultaneous detection of several respiratory pathogens in real time. The results are reliable and give the clinician a picture of the current epidemiological situation, thus minimising guesswork.     

Prevalence of respiratory viruses, including newly identified viruses, in hospitalised children in Austria

The aim of this epidemiological study was to determine the prevalence of respiratory viruses, including new viruses, in hospitalised children in Austria. Two hundred fourteen nasopharyngeal samples from hospitalised children were tested for the presence of viruses using cell culture and PCR and/or viral antigen assays. The results revealed a parainfluenza virus 1 (PIV1) outbreak that ended right before the onset of the influenza season, with nearly no overlapping, moderate respiratory syncytial virus (RSV) activity, and only a few adenoviruses. Human metapneumovirus (hMPV) was present in 14.5% of the total samples but was detected in combination with other viruses in only five cases: with PIV1 in three cases and with RSV in two cases. There were no cases of dual infection with hMPV and flu or adenovirus. This suggests that hMPV alone is a leading cause of hospitalisation in children under 1 year of age. Interestingly, hMPV, in contrast to RSV, coincided with PIV1 but was absent during the community outbreak of the flu. Samples were also tested for Mimiviridae, a group of newly described DNA viruses that are similar to Legionella spp., replicate in water amoebae, and also have been found in alveolar cells. However, mimivirus was detected neither in respiratory samples nor in amoebae-containing water samples, indicating that this particular type of virus is either not abundant or does not contribute to paediatric respiratory illnesses.     

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。     

Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.     

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.     

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.     

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.