Real-Time Detection of Influenza A, Influenza B, and Respiratory Syncytial Virus A and B in Respiratory Specimens by Use of Nanoparticle Probes

Specimens.A total of 720 nasopharyngeal swabs were collected from two geographically diverse sites, one in the southern United States and another in the southeastern United States, during the 2007 to 2008 and 2008 to 2009 respiratory virus seasons. Subjects had a distribution of ages ranging from less than 1 year to over 65 years. All specimens were tested for influenza virus and RSV at the time of collection using culture or virus-specific DFA detection. Residual specimens were stored at the time of initial analysis at −70°C for later analysis using the VRNAT or RVNAT_SP at one of three clinical test sites. This study has been reviewed and approved by each site's institutional review board.

Equipment and instrumentation.The VRNAT_SP requires the use of the Verigene SP system. The Verigene SP comprises a single Verigene reader (approximately 16 in. by 12 in.), which has the capability to read and interpret the VRNAT_SP microarray slides in <1 min. The reader can interface with up to 32 Verigene SP processors. Each processor is free standing (approximately 8 in. by 23 in.) and is capable of random-access, multifunctional test processing, including nucleic acid extraction, reverse transcription (if necessary), target amplification (if necessary), and DNA hybridization. The system can accommodate several different test cartridges for additional nucleic acid tests available from the manufacturer.

Manual extraction and amplification for the semiautomated VRNAT.Residual specimen samples were thawed to room temperature, and a 200-μl aliquot was removed. A 50-μl aliquot of MS2 phage was added to the clinical specimen as an internal control for extraction and PCR inhibition. Nucleic acid was extracted from the entire 250 μl using the NucliSENS EasyMAG (bioMerieux, Marcy l'Etoile, France) and was eluted into a final volume of 60 μl buffer. RT-PCR was carried out by adding 5 μl of the eluate to 20 μl of an enzyme premix containing Tfi SuperMix (Invitrogen, Carlsbad, CA), uracil deglycosylase (UDG), reverse transcriptase, and proprietary oligonucleotide primers specific for each viral target. Following amplification, 10 μl of the product was mixed with sample buffer and loaded into a disposable RV test cartridge. Processing and hybridization were carried out by the Verigene processor. Upon completion of processing, the RV test cartridge was removed from the processor and analyzed using the Verigene reader. Results were reported as “detected” or “not detected” for each of the four reportable agents detected by this assay (influenza A virus, influenza B virus, RSV A, and RSV B). The VRNAT is capable of discriminating RSV A and RSV B; however, the initial characterization by culture/DFA did not distinguish the two subtypes, so VRNAT results are reported as “RSV detected” when either subtype (RSV A or RSV B) is detected. A message of “no call” was reported by the reader in the event of assay failure, such as an internal control failure. Samples generating this message were rerun to obtain a valid result.

Specimen preparation and loading for the fully automated RVNAT_SP assay.Single-use extraction and amplification trays containing all necessary reagents were loaded into the Verigene SP processor. Specimens were thawed, and a 200-μl aliquot was transferred to the specimen well in the extraction tray. A single-use RV test cartridge containing the slide array and hybridization reagents was loaded into the Verigene SP processor, and the assay was started. Upon completion of the test, the RV test cartridge was removed from the processor and the hybridization slide was inserted into the Verigene reader. Results were recorded as described above.

Analysis of discrepant specimens.Specimens producing discrepant results between culture/DFA and VRNAT were resolved using bidirectional sequencing employing primer annealing sites that were different from that used in the VRNAT assay. Approximately 100 to 200 ng of purified DNA was sent to ACGT, Inc. (Wheeling, IL) for sequencing. All amplification reactions were performed on a Bio-Rad (Hercules, CA) C1000 thermal cycler or GeneAmp (Applied Biosystems, Carlsbad, CA) PCR System 9700 thermal cycler. Four positive controls [pGEM 3Zf(+) plasmid DNA with M13F (−21) primer] and four negative controls [nuclease-free water with M13F (−21) primer] were included in a 96-well plate and run concomitantly with the samples. The sequencing reaction products were purified with the Agencourt CleanSeq kit (Beckman Coulter, Danvers, MA) and analyzed by the ABI 3730 XL genetic analyzer (Applied Biosystems, Carlsbad, CA). The raw data from sequencing reactions were collected using ABI 3730 foundation data collection software and processed by InterPhace software (CodonCode, Dedham, MA) to generate a Phred quality score for each base in the sequence. The sequence data were used for analysis if the controls met the following criteria: none of the negative controls (0/4) have more than 100 consecutive bases with a score of Phred 30 (99.9% accuracy) or above, while 50% of the positive controls (2/4), at a minimum, have at least 200 bases with scores of Phred 30 (99.9% accuracy) or above. Sequencing data were assembled by Sequencher (version 4.2) software (GeneCodes, Ann Arbor, MI) to generate a single contig for each PCR product. A consensus sequence was produced from each alignment, which was then compared to those of the reference strains (see Table S1 in the supplemental material) for confirmation of virus identification.

Analytical specificity experiments.A total of 15 viral and 23 bacterial strains available from ATCC (see Table S2 in the supplemental material) were obtained, and stock dilution cultures were made. Bacterial CFU/ml was determined by serial dilution. The viral strains were quantified by assessing tissue culture infectivity to provide 50% tissue culture infective doses (TCID₅₀)/ml. The TCID₅₀/ml value was multiplied by a factor of 0.7 to obtain an approximate PFU/ml. Pure suspensions of each agent were extracted, amplified, and analyzed using the VRNAT, under conditions identical to those used for the analysis of clinical specimens.

Analytical sensitivity experiments.A stock culture containing a defined PFU/ml of each virus was diluted serially into universal transport media (Copan, Murrieta, CA). Dilutions of each agent were tested a minimum of three times using the VRNAT or RVNAT_SP to establish a limit of detection (LoD). The presumptive LoD was then confirmed by an additional 20 replicate experiments. Achievement of ≥95% positive results was required to define the LoD for each agent.

Statistical analysis.The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined using standard methods (22). For calculations made before resolution of discrepant results, a true-positive/negative result was based solely on culture/DFA regardless of VRNAT result. Following discrepancy resolution, a true positive was defined by agreement between culture/DFA and VRNAT or by positivity in either assay, which was confirmed using bidirectional sequencing.

Analytical specificity/sensitivity and cross-reactivity with other infectious agents.To demonstrate the analytical specificity and sensitivity of the VRNAT, established titers of 11 different strains of influenza virus or RSV were serially diluted into viral transport medium and analyzed using the VRNAT. The limit of detection (LoD) for each strain, defined as the lowest viral concentration at which ≥95% of the replicates tested positive, was established and confirmed by not less than 20 replicate assays. Specifically, the LoD of influenza A and B virus strains tested ranged from 2 to 60 TCID₅₀/ml (Table 1). This is well below the established level of 10³ to 10⁷ TCID₅₀/ml of influenza A virus shed in the nasopharynx during active infections (36). Of special interest, the VRNAT was able to detect the novel 2009 H1N1 swine origin influenza A virus subtype at a similarly low LoD of 50 TCID₅₀/ml (Table 1). To assess the analytical specificity of the assay, a comprehensive panel of common respiratory viruses (15) and bacteria (23) was examined. Among the viruses tested were human adenovirus types 1 and 7, two types of human coronavirus, cytomegalovirus, an enterovirus (coxsackievirus B4), Epstein-Barr virus, human parainfluenza types 1, 2, 3, and 4a, measles virus, mumps virus, and human metapneumovirus. Included in the bacterial pathogens were common strains of Acinetobacter, Bordetella, Chlamydophila, Corynebacterium, Escherichia coli, Haemophilus, Klebsiella, Lactobacillus, Legionella, Listeria, Moraxella, Neisseria, Proteus, Pseudomonas, Staphylococcus, Streptococcus, Mycoplasma, and Mycobacterium. All strains were processed in pure culture by the VRNAT at concentrations ranging from 10⁵ to 10⁷ CFU/ml or PFU/ml (see Table S2 in the supplemental material). None of the tested microbes produced a positive result, demonstrating 100% specificity.

Comparison of VRNAT to culture/DFA methods for the detection of influenza A virus, influenza B virus, and RSV A and B.A total of 720 nasopharyngeal specimens were collected prospectively from multiple sites during the 2007 to 2008 and 2008 to 2009 respiratory virus seasons. The patient population was chosen to include a broad distribution of ages, containing 120 subjects 0 to 1 year of age, 229 between the ages of 1 and 5 years, 129 between the ages of 5 and 20 years, 204 between the ages of 20 and 65 years, and 38 over the age of 65 years. Patient specimens were assayed for the presence of influenza A virus, influenza B virus, RSV A, and RSV B using culture/DFA methods at the time of collection. Of the 720 specimens analyzed by culture/DFA, 123 (17%) were positive for influenza A virus by culture/DFA, 31 (4%) were positive for influenza B virus, 49 (7%) were positive for RSV, and 517 (72%) specimens were culture negative. When the same specimens were analyzed using the VRNAT, 181 (25%) were positive for influenza A virus, 40 (6%) were positive for influenza B virus, and 101 (14%) were positive for RSV A or B (Table 2). In comparison to culture/DFA, these results translate to VRNAT sensitivity and specificity of 96.6% and 93.6%, respectively (Table 3). Of the 133 discrepant specimens between the VRNAT and culture, bidirectional sequencing revealed that the VRNAT detected 108 additional positives (58 influenza A virus, 4 influenza B virus, and 46 RSV), all of which were negative by culture/DFA. Following discrepancy resolution, the VRNAT sensitivity for all agents was 98.0% and specificity was 96.5%. This also resulted in an overall increase in the PPV of the VRNAT from 60.9% to 91.6% (Table 3). Of note, since sequencing requires a significantly greater amount of amplified product than is needed for detection by the VRNAT, a few of the discrepant samples that were positive by VRNAT could not be confirmed by sequencing and may have been the result of low-level infections. Among the clinical specimens, there were also a number of dual infections comprising different influenza virus types or influenza virus-RSV coinfections. This included 3 influenza A virus-influenza B virus coinfections, 5 influenza A virus-RSV A or B coinfections, 4 influenza B virus-RSV A or B coinfections, and 3 RSV A-RSV B coinfections. The agents in these coinfections were reliably detected by the VRNAT, demonstrating its ability to simultaneously detect multiple pathogens from a single specimen.

Comparison of VRNAT to RVNAT_SP.In a follow-up study, we compared the performance of the fully automated RVNAT_SP to that of the semiautomated VRNAT previously established in this study. To accomplish this, a total of 558 clinical specimens, including a distribution of culture/DFA-positive influenza A and B virus and RSV specimens, were analyzed using both the VRNAT and RVNAT_SP. Of the 558 specimens, 195 were positive for influenza A or B virus or RSV using the VRNAT and 191 were positive using the RVNAT_SP. This resulted in a concordance of 99.3% between the two assays (Table 4). All 4 specimens that were initially positive by VRNAT and negative by RVNAT_SP were positive by the RVNAT_SP following a repeat test. These results demonstrate the equivalence of the two systems for detection of influenza A virus, influenza B virus, and RSV A and B from clinical specimens.

Analytical sensitivity experiments were conducted independently using the RVNAT_SP. The experimental setup was identical to that used to establish the analytical sensitivity of the VRNAT (see results above and Materials and Methods), and experiments were conducted using one strain each of influenza A virus, influenza B virus, RSV A, and RSV B. Results indicated similarly high sensitivities for all agents tested, ranging from 2 to 50 TCID₅₀/ml (Table 1).