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J Clin Microbiol. 2012 July; 50(7): 2282–2288.
PMCID: PMC3405573

Comparison of the Luminex xTAG RVP Fast Assay and the Idaho Technology FilmArray RP Assay for Detection of Respiratory Viruses in Pediatric Patients at a Cancer Hospital

Abstract

Respiratory viruses are increasingly recognized as serious causes of morbidity and mortality in immunocompromised patients. The rapid and sensitive detection of respiratory viruses is essential for the early diagnosis and administration of appropriate antiviral therapy, as well as for the effective implementation of infection control measures. We compared the performance of two commercial assays, xTAG RVP Fast (Luminex Diagnostics, Toronto, Canada) and FilmArray RVP (FA RVP; Idaho Technology, Salt Lake City, UT), in pediatric patients at Memorial Sloan-Kettering Cancer Center. These assays detect the following viruses: respiratory syncytial virus; influenza A and B viruses; parainfluenza viruses 1, 2, 3, and 4; human metapneumovirus; adenovirus; enterovirus-rhinovirus; coronaviruses NL63, HKU1, 229E, and OC43; and bocavirus. We tested a total of 358 respiratory specimens from 173 pediatric patients previously tested by direct fluorescence assay (DFA) and viral culture. The overall detection rate (number of positive specimens/total specimens) for viruses tested by all methods was 24% for DFA/culture, 45% for xTAG RVP Fast, and 51% for FA RVP. The agreement between the two multiplex assays was 84.5%, and the difference in detection rate was statistically significant (P < 0.0001). Overall, the FA RVP assay was more sensitive than the xTAG RVP Fast assay and had a turnaround time of approximately 1 h. The sensitivity, simplicity, and random-access platform make FA RVP an excellent choice for laboratory on-demand service with low to medium volume.

INTRODUCTION

Infection due to community-acquired respiratory viruses is associated with significant morbidity and mortality among patients with hematologic malignancy and those undergoing hematopoietic stem cell transplant (HSCT) (11). In the last decade, the long-term implications of respiratory virus infection among susceptible hosts have increasingly been recognized. In addition to the immediate morbidity and mortality associated with the infection, long-term decline in pulmonary function has been demonstrated in survivors of lung transplant and HSCT (25, 7, 11). In children undergoing treatment for hematologic malignancies, infections due to influenza viruses, parainfluenza viruses, and respiratory syncytial virus are particularly common and have significant impact on oncologic care (12, 23, 24). The clinical significance of other viruses, such as bocavirus, human coronaviruses (HCoVs; NL63 and HKU1), parainfluenza virus 4 (PIV4), and rhinovirus, is less clear (21). Mixed infections with two or more respiratory viruses are common in children but are not easily detected by conventional methods, hence the biological significance of dual infections currently is not well understood (9, 12).

The rapid and sensitive detection of respiratory viruses in the clinical microbiology laboratory is essential for early and accurate diagnosis, the timely administration of appropriate antiviral therapy, and infection control measures, such as the institution of droplet precautions to prevent nosocomial transmission. However, conventional methods have several limitations: viral culture is time- and labor-intensive, and direct fluorescent antibody assay (DFA) and immunochromatographic antigen testing, although rapid, have poor sensitivity for the detection of most viruses. Furthermore, these methods have a limited range of detection and can be subjective, relying on technical expertise for the interpretation of cytopathic effect (CPE) in cell culture (8).

Molecular assays, based on PCR, are rapid and sensitive compared to conventional methods (14, 15). Commercial assays, both research use only (RUO) and Food and Drug Administration (FDA) cleared, are widely available, facilitating the implementation of these technologies in the diagnostic microbiology laboratory.

One of the first FDA-cleared multiplexed molecular assays, the xTAG respiratory viral panel (RVP) assay (Luminex Molecular Diagnostics, Toronto, Canada), targets 12 viruses and subtypes (respiratory syncytial viruses [RSV] A and B, influenza A virus [Flu A] [H1 subtype, H3 subtype, and untypeable], influenza B virus [Flu B]1, PIV1 to PIV3, human metapneumovirus [hMPV], adenovirus, and enterovirus/rhinovirus). In 2011, the FilmArray respiratory viral panel (FA RVP) assay (Idaho Technology, Salt Lake City, UT) received FDA clearance for the detection of 15 viruses and subtypes, including the virus targets in xTAG RVP plus human coronaviruses (NL63 and HKU1) and PIV4. At the time of our study, RUO versions of the xTAG RVP assay, xTAG RVP Fast, and FA RVP were under consideration for FDA clearance. These two assays included all of the viral targets in xTAG RVP plus human coronaviruses (NL63, HKU1, 229E, and OC43), bocavirus, and PIV4. FA RVP also included three bacterial targets: Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

We conducted a study to compare the performance of xTAG RVP Fast and FA RVP for the detection of clinically significant viruses among pediatric patients at the Memorial Sloan-Kettering Cancer Center (MSKCC). In addition to assay performance, we compared other performance characteristics, including workflow, reliability, throughput, turnaround time, and direct costs.

MATERIALS AND METHODS

Patients.

This was a retrospective study (July 2010 to March 2011) conducted on banked specimens collected from pediatric patients (0 to 21 years old) with upper and lower respiratory tract infections. Upper respiratory tract infection was defined as the presence of rhinorrhea, pharyngitis, otitis, and sinusitis or sinus congestion. Lower respiratory tract infection was defined as the presence of cough, wheezing, dyspnea, or abnormal lung findings (i.e., the presence of infiltrates on imaging). Clinical data on symptoms, underlying diseases, and demographic information were extracted from the review of patient charts. The study was approved by the MSKCC Institutional Review Board, and HIPAA waiver of the authorization was granted.

Clinical specimens.

Three hundred fifty-eight respiratory specimens were tested in this study. From July 2010 to January 2011, 303 consecutive samples received in the laboratory from pediatric patients were tested. These included nasopharyngeal swabs (NP; n = 280), bronchial washings and lavages (BW/BL; n = 8), throat swabs (TS; n = 13), and sputum (SPEX; 2). One ml of each specimen was stored at −70°C until further testing. As the community activity of certain viruses was not widespread between July 2010 and January 2011, an additional 55 known positive samples (all NP), collected between January 2011 and March 2011, were included to determine the performance of the assays for these common viruses, bringing the total number of tested specimens to 358.

DFA test.

DFA tests were performed on all specimen types except throat swabs. Reagents used for the DFA tests included the D3UItra DFA respiratory virus ID kit and the D3 DFA metapneumovirus ID kit (Diagnostic Hybrids, Athens, OH). Both tests were performed according to the manufacturers' instructions.

Viral culture.

Viral culture was performed using established methods (6). All swabs were collected using flocked swabs (FLoQwabs; Copan, Murrieta, CA) in M4RT transport medium (Remel, Lenexa, KS). Each specimen (0.2 ml) was inoculated into three cells lines, a primary rhesus monkey kidney (RMK; Viromed, Minnetonka, MN) and two human lung diploid fibroblast cell lines (MRC-5; Viromed, Minnetonka, MN; and WI-38; Diagnostic Hybrids, Athens, OH), which were incubated at 37°C (RMK and MRC-5) and 33°C (WI-38). Cultures were read daily for the detection of CPE during the first 7 days of incubation and every other day until day 14. For hMPV, a shell vial was set up in LLC-MK2 cells and read after 48 h. CPE observed in viral culture were confirmed by DFA as described above, except for rhinoviruses, which were detected by characteristic CPE and reported as rhinovirus-like for lack of available confirmatory testing by DFA.

Nucleic acid extraction.

Total nucleic acid extraction was performed on a MagNA Pure LC (Roche Diagnostics, Indianapolis, IN). Two hundred μl of specimen and an external positive control, influenza virus H1N1 2009 (Zeptometrix Corporation, Buffalo, NY), were transferred into a well on the MagNA Pure sample cartridge. Twenty μl of xTAG MS2 (internal control) was added to each specimen, and the cartridge was placed on the MagNA Pure instrument. Specimens were extracted using a Total nucleic acid extraction kit (Roche Diagnostics, Indianapolis, IN) and the Total NA variable-elution-volume protocol with a sample input of 200 μl and an elution volume of 55 μl.

Multiplex RT-PCR.

Reverse transcriptase PCR (RT-PCR) was performed according to the xTAG RVP Fast assay product insert instructions. PCR amplification was performed on a MyCycler thermocycler (Bio-Rad, Hercules, CA) using the following cycling parameters: 1 preheating step at 50°C for 20 min; 1 template denaturing cycle at 95°C for 15 min; 34 amplification cycles at 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s; and ending with 1 cycle at 72°C for 2 min and a hold at 4°C until ready for use.

Hybridization and detection.

All reagents, the xTAG RVP Fast bead mix, the reporter buffer, and the xTAG streptavidin and phycoerythrin G15 (SA-PE) conjugate were vortexed before use. A 1:75 dilution of SA-PE G15 was prepared in xTAG reporter buffer. Each hybridization reaction mixture contained 20 μl of bead mix, 2 μl of amplified DNA, and 75 μl of SA-PE G15 on a 96-well plate. The plate was incubated for 20 min at 45°C, followed by analysis on a Luminex 200 instrument (LMD, Toronto, Canada) using the xTAG RVP Fast (IUO) 20-plex software.

RVP FA assay.

The extraction, amplification, and detection steps take place in different chambers of a self-contained, single-use pouch containing all of the reagents necessary for the detection of the 15 respiratory virus targets. Additional reagents and disposables included a hydration syringe and a sample loading syringe, a hydration solution vial, and a sample buffer vial. The RVP FilmArray test was performed according to the manufacturer's instructions. Briefly, 1 ml of hydration solution was added to the pouch using the hydration syringe. Using a transfer pipette, approximately 300 μl of respiratory sample was added to the sample buffer vial, and the resulting mixture was transferred to the pouch using the sample loading syringe. The pouch was then placed on the FilmArray instrument and the test performed using the FilmArray operational software.

Discordant analysis.

The gold standard used to determine true-positive and false-negative results was a combination gold standard (i.e., true positive was a specimen that was positive by at least two methods). Any specimen that was positive by only one of the molecular assays was further resolved by a review of medical records to determine if the patient had a previous positive for the same virus (by DFA and/or culture or by both molecular assays) on the most recent specimen tested (within 8 weeks). For samples which could not be resolved by a review of medical records, additional testing was performed using the Resplex II assay (Qiagen, Germantown, MD). The Resplex II is a multiplex assay that detects the 15 viruses and subtypes common to both xTAG RVP Fast and FA RVP. The testing was performed at the Molecular Infectious Diseases Laboratory at Vanderbilt University as previously reported (13).

Statistical analysis.

Statistical analysis was performed using Fisher's exact test. P < 0.05 was considered significant.

RESULTS

Clinical presentation.

Three hundred fifty-eight specimens tested during the study period were obtained from 173 patients. The clinical characteristics of the patients are listed in Table 1. The most common underlying disease in the study cohort was hematologic malignancies (46.8%), mostly acute leukemia (85%). Upper respiratory tract infection (URI) symptoms were present in 71.6% of patients (124/173) and lower respiratory tract infection (LRI) symptoms in 63.6% of patients (110/173). Thirty-nine percent of patients (43/110) with LRI had radiographic imaging performed, and infiltrates were seen in 39.5% of patients (17/43). Fever (temperature, ≥38°C) was present in 48.6% (84/173) of patients, and 24.3% (42/173) were neutropenic (absolute neutrophil count, <500 cells/ml) at the time of testing. Fourteen patients (8%) did not have any respiratory symptoms.

Table 1
Patient characteristics

Discordant analysis.

There were a total of 47 specimens that were positive by only one of the two PCR assays (11 were positive only by xTAG RVP Fast, and 36 were positive only by FA RVP). However, because several specimens had multiple viruses, a total of 73 specimens were discordant (i.e., detection of one virus by both assays and detection of an additional virus by only one of the assays). In general, FA RVP detected more viruses than xTAG RVP (223 versus 180), except for rhinoviruses/enteroviruses and PIV4 (Table 2). Forty-three of the 73 discordant results were resolved by the results of the DFA/culture (10/43) or by a review of medical records (33/43) as described in Materials and Methods. The remaining specimens (30/73) were tested by Resplex II assay as shown in Table 3.

Table 2
Comparison of DFA, culture, xTAG RVP FAST, and FA RVP detection of respiratory viruses in pediatric patient specimensd
Table 3
Resolution of discordant results

Consecutive specimens (July 2010 to January 2011).

A total of 303 consecutive respiratory specimens were obtained between July 2010 and January 2011 from 153 pediatric patients; these specimens were tested by DFA/culture, xTAG RVP Fast, and FA RVP. Sixty-five patients had two or more specimens tested during the study period. The detection rate (number of positive specimens/total number of specimens) for each method was 24.1% (73/303) for DFA/culture, 53.1% (161/303) for xTAG RVP Fast, and 61.4% (186/303) for FA RVP. Considering viruses common to all detection methods, the detection rate was 24.1% for DFA/culture (73/303), 45% for xTAG RVP Fast (136/303), and 51% for FA RVP (154/303). Coinfections were detected as 3 episodes (in 2 patients) by culture, 14 episodes (in 12 patients) by xTAG RVP Fast, and 24 episodes (in 20 patients) by FA RVP. Triple infections were detected in two patients by xTAG RVP Fast and seven patients by FA RVP (including the two detected by xTAG RVP Fast). The total number of isolates identified by xTAG RVP Fast and FA RVP was 180 isolates in 96 specimens and 223 isolates in 105 specimens, respectively. Following the resolution of discordant results, the total number of confirmed isolates detected by xTAG RVP Fast and FA RVP was 172 and 197 isolates, respectively. A total of 14 double infections and 2 triple infections detected by xTAG RVP Fast were confirmed by the gold standard. For FA RVP, 15 double infections and 3 triple infections were confirmed by the gold standard. The agreement between the two multiplex assays was 84.5% (kappa score, 0.685; good agreement). The difference in overall detection rate between xTAG RVP Fast and FA RVP (172 versus 197 isolates) was statistically significant (P < 0.0001).

Assay comparison for specific viruses.

A head-to-head comparison of individual target detection between xTAG RVP Fast and FA RVP reveals some significant differences. Results are summarized in Table 2 and are described individually below.

Flu A and B.

The detection of influenza A virus (Flu A) was similar for both xTAG RVP Fast and FA RVP, with both assays detecting additional Flu A isolates not detected by DFA/culture. xTAG RVP Fast did not detect one of two Flu A 2009 H1 samples detected by both DFA/culture and FA RVP, and it misidentified a Flu B sample as Flu A H-3 (both DFA/culture and FA RVP identified it as Flu B). The overall sensitivity and specificity of xTAG RVP Fast for Flu A was 95.2 and 99.6%, respectively, and the sensitivity and specificity of FA RVP was 100%. Two influenza B viruses were identified in culture among the study isolates; xTAG RVP Fast detected one of two isolates, while FA RVP detected both.

RSV.

A greater difference between xTAG and FA RVP was observed in RSV detection. The sensitivity and specificity of xTAG RVP Fast for RSV was 60 and 100%, respectively, while the sensitivity of FA RVP for RSV was 100%, with a specificity of 98.5%. Of note, xTAG RVP Fast had lower sensitivity for the detection of RSV (12 isolates in 8 patients) than did DFA/culture (16 isolates in 10 patients).

Bocavirus.

Bocavirus was detected in eight samples from five patients. Four of 8 positive samples were serially obtained from a single patient during a 2-month period, with the first specimen being positive by both xTAG RVP Fast and the following three positive by FA RVP only. Of the remaining 4 unique specimens, 2 were positive by both assays and 2 were considered false positive by FA RVP, as they were not confirmed by the Resplex II assay. The sensitivity of xTAG RVP Fast for bocavirus was 50% with a specificity of 100%, while the FA RVP had a sensitivity of 100% with a specificity of 99.3%.

HCoV.

The coronavirus most frequently detected by both xTAG RVP Fast and FA RVP was HCoV NL63, and neither assay detected HCoV 229E. Overall, FA RVP detected 42 coronaviruses in 35 patients, while xTAG RVP Fast detected 26 coronaviruses in 14 patients. The sensitivity and specificity of xTAG RVP Fast was 92.8 and 100% for HCoV C43, 33.3 and 100% for HCoV HKU1, and 85.7 and 100% for HCoV NL63, respectively. The sensitivity and specificity of FA RVP was 100 and 98.6% for HCoV OC43, 100 and 100% for HCoV HKU1, and 100 and 98.6% for HCoV NL63, respectively.

PIV.

The number of patients positive for PIV during the study period was small. In general, xTAG RVP Fast detected fewer PIV1, PIV2, and PIV3 samples than both DFA/culture and FA RVP. The specificity of xTAG RVP was 100% for PIV1, PIV2, and PIV3, with a sensitivity of 33.3% for PIV1, 36.3% for PIV2, and 72.7% for PIV3. FA RVP had a sensitivity of 100% for PIV1, PIV2, and PIV3, with a specificity of 100% for PIV1 and PIV3 and 99.6% for PIV2. PIV4 was detected more frequently by xTAG RVP Fast (7 cases in 6 patients) than FA RVP (2 cases in 2 patients), with a sensitivity of 100% and a specificity of 98.9% for xTAG RVP and a sensitivity of 50% and a specificity of 100% for FA RVP.

Rhinovirus/enterovirus.

Both assays used in the study were unable to differentiate between rhinovirus and enterovirus. Both assays detected significantly more rhinoviruses/enteroviruses than culture; xTAG RVP Fast detected 6 more cases than FA RVP. The sensitivity and specificity of xTAG RVP for rhinovirus/enterovirus was 97.6 and 97.7%, respectively, while the sensitivity and specificity of FA RVP for rhinovirus/enterovirus was 91.6 and 98.5%, respectively.

Adenovirus and hMPV.

Only a single case each of adenovirus and hMPV were detected among the study cohort. Both assays (xTAG RVP Fast and FA RVP) identified the viruses, which were not detected by DFA/culture.

Testing of specimens positive by DFA/culture (January 2011 to March 2011).

Fifty-five known positive nasopharyngeal swabs specimens from 42 pediatric patients were tested by xTAG RVP Fast and FA RVP. These specimens were included in the study to increase the number of samples positive for Flu A (n = 14) and Flu B (n = 23), RSV (n = 14), and hMPV (n = 2), as these infections were not widespread in the community during the study period. The agreement between xTAG RVP Fast and FA RVP was 100% for Flu A, hMPV, and PIV3, with FA RVP specifically identifying 2009 H1N1 Flu A and xTAG RVP Fast reporting it as untypeable Flu A (Table 4). Previous reports have shown that Flu A that is untypeable by xTAG RVP is indeed 2009 H1N1 Flu A (9). For Flu B, xTAG RVP Fast detected 13/23 (56%) isolates while FA RVP detected all 23 Flu B samples with two coinfections. Both xTAG RVP Fast and FA RVP detected additional viruses (multiple infections) not detected by DFA or culture, mainly rhinoviruses and coronaviruses.

Table 4
Results of 55 nasopharyngeal swabs known to be positive by DFA/culture

Workflow and cost.

Table 5 summarizes the workflow characteristics and cost of each assay at our institution. FA RVP pouches are available in boxes of 30 tests, with pricing listed at $3,870 for a cost of $129/pouch. All controls for the assay are internal to the pouch. One kit of xTAG RVP Fast contains reagents for 96 tests. Additionally, external controls need to be included in each run, and for a full plate, more than one control is recommended. Reagents for the extraction step are purchased separately. With the inclusion of costs for NA extraction using the MagNA pure instrument ($6.75/sample), the overall cost was $113.13 per assay. The hands-on time for the FA RVP assay is ~5 min/specimen, with a run time on the instrument of 1 h for one specimen. The hands-on time for xTAG RVP Fast is 60 to 80 min for 24 tests (21 patients and 3 controls), with 30 to 40 min for extraction setup, 15 to 20 min for PCR setup, and 15 to 20 min for hybridization setup. The overall duration of the assay is 5 to 6 h, with an average of 90 min for extraction, 150 min for PCR, 45 min for hybridization, and 10 to 15 min for reading on the Luminex instrument. It would take 26 h (65 min per specimen) to perform 24 FA RVP tests on one FA instrument.

Table 5
Comparison of workflow and cost of each assay

Although xTAG RVP Fast requires greater technical skills than FA RVP, the number of specimens requiring repeat testing was similar between the two tests: 9 internal control failures for xTAG RVP Fast and 6 invalid results for FA RVP (Table 5). All failed, and invalid results were repeated and yielded interpretable results on repeat.

DISCUSSION

In this study, we compared the performance characteristics of two commercial multiplex assays for the detection of respiratory viruses. The most common viruses that are considered significant respiratory pathogens in immunocompromised hosts include influenza A virus, influenza B virus, RSV, PIV1 to PIV3, hMPV, and picornaviruses (rhinoviruses and enteroviruses) (11). Newly discovered viruses, including bocavirus, PIV4, novel coronaviruses, and rhinoviruses, are increasingly being recognized as causes of respiratory illness in this population. However, the ability to detect these novel viruses as well as other significant pathogens by conventional virologic methods is limited.

Molecular assays have been shown to be superior to traditional methods for virus detection and further allow the identification of novel viruses, most of which are not cultivable by techniques routinely used in diagnostic laboratories (15). In our study, xTAG RVP Fast and FA RVP had significantly higher sensitivities for the detection of respiratory viruses than traditional methods, such as DFA and viral culture. The sensitivity of FA RVP was generally higher than that of xTAG RVP Fast, which translated as a lower specificity, since the additional viruses not detected by all other methods could be considered false positives. The difference in sensitivity between the two assays remained statistically significant when only confirmed positive isolates were considered. The increased sensitivity of the FA RVP assay might be due to the incorporation of nested PCR as the amplification method in the FA RVP assay, increasing the likelihood of the detection of a low viral burden. Additionally, there is more than one reaction site in each pouch for the second-stage PCR for each virus, further increasing the sensitivity of the assay. The sensitivity of the FA RVP was closer to that of the Resplex II assay, in which a novel template-enhanced multiplexing process was used for amplification (13). In a previous study by Rand and colleagues, FA RVP was also shown to detect more total viruses than the xTAG RVP assay (20). In persons with cancer, where a positive nasopharyngeal swab for respiratory viruses has substantial impact on oncologic care and outcome, the implementation of tests with increased sensitivity is imperative.

The lower sensitivity of xTAG RVP Fast was especially noted for Flu B and RSV. A previous report by Pabbaraju et al. also noted a decreased sensitivity for Flu B in a study comparing xTAG RVP Fast to xTAG RVP (18). As the authors of that study suggested, sequence variation in the hemagglutinin gene of Flu B could result in primer mismatch and lower sensitivity. Recent mutations in RSV have resulted in the lower detection of the virus with the current FDA-cleared version of the xTAG assay during the 2010 to 2011 respiratory season (R. T. Horvat, personal communication). Although the primers and targets are different in the xTAG RVP Fast version (per the manufacturer), the lower sensitivity of the assay for RSV detection compared to that of FA RVP and DFA could similarly be attributed to the genetic evolution of the virus. The detection rate for Flu A was similar in both assays, with the added advantage of the specific detection of the 2009 H1N1 Flu A by FA RVP as opposed to being labeled an untypeable result, as was the case for xTAG RVP Fast. This can have important clinical implications in outbreak settings and in the selection of antiviral therapy for treatment as various susceptibilities in circulating influenza virus strains occur.

The detection of PIV was higher with FA RVP than xTAG RVP Fast. For PIV3, FA RVP was sensitive enough to detect the prolonged shedding of the virus in one patient for an additional month after DFA, culture, and xTAG RVP became negative. This discrepancy could have been due to low viral load, as suggested in a recent study comparing FA RVP to xTAG RVP. Rand et al. showed that most of the FA RVP-positive, xTAG RVP-negative specimens for RSV had high cycle threshold values (20). Although no cycle values were available for our results, a similar explanation could apply to our results for PIV3 and RSV. This is particularly important specifically for the detection of PIV and RSV, where the asymptomatic shedding of virus is known to be the source of several outbreaks and the detection of low viral burden is likely to have a clinical impact (10, 17, 19).

Both assays detected a higher number of rhinovirus/enterovirus infections and several coronaviruses not routinely detected by DFA or culture. This result is similar to that reported by Rand et al. for rhinovirus detection by FA RVP and xTAG RVP (20). Their comparison did not include coronaviruses, which are not detected by the xTAG RVP assay. Approximately 30% of pediatric patients were positive for rhinovirus (20%) and/or coronavirus (10%). The detection rate of these two viruses by molecular assays was twice that of culture. Similar infection rates have been reported previously among pediatric patients with leukemia and allogeneic HSCT recipients, where infection often occurs in the first 100 days after transplant (12, 16). As persons with cancer and HSCT recipients can develop devastating complications from respiratory virus infections, the timely and sensitive diagnosis of these viruses is particularly important to develop a better understanding of their clinical impact and to develop effective treatment and control strategies.

Human bocavirus was included in both RVP assays. Bocavirus is a recently identified parvovirus (1), and its clinical significance as a respiratory pathogen has not been fully elucidated (22). In our study, 3 patients were infected with confirmed bocavirus, and 2 of these patients had dual infections with rhinovirus, coronavirus, and Flu B. Although more than 90% of our pediatric patients were symptomatic (Table 1), including the cases with bocavirus, the presence of additional coinfecting viruses makes it challenging to ascertain the role of bocavirus as a sole respiratory pathogen.

With the implementation of rapid and highly sensitive molecular assays, one of the biggest impacts will be on infection control practices. Currently at MSKCC, patients are placed on droplet precautions until they are asymptomatic and the DFA and culture results are negative (minimum isolation duration is 3 days irrespective of results). With the implementation of PCR, diagnostic yield in symptomatic patients being tested for viral infection will increase. A large number of patients will be found positive for viruses other than the usual suspects (i.e., influenza virus and RSV), infections will likely be detected early, and prolonged shedding will be commonly encountered. Shedding for up to 3 months has been described in 13% of HSCT recipients with rhinovirus and coronavirus infection (16). The replication competence of viruses among prolonged shedders is often debated, and the best infection control practice in this situation has not been determined. While the overall impact of the transition to PCR-based testing is favorable, some practical challenges will arise in the process.

In conclusion, each assay evaluated in this study had advantages and disadvantages, but overall the performance of FA RVP was superior to that of xTAG RVP Fast for the majority of viruses in the panels. In addition to detecting almost two to three times as many viruses in our pediatric population than DFA/culture, these two assays significantly cut down on the turnaround time for results from several days (e.g., 2 to 3 days for Flu A and up to 7 days for rhinoviruses) to a few hours for FA RVP and up to 24 h for xTAG RVP Fast. Several reports have established the superiority of molecular assays for respiratory virus detection; however, to our knowledge, this study is the first to compare the RUO version of these two assays. FA RVP was FDA cleared in May 2011 for the detection of 15 viruses and subtypes, including influenza A virus, influenza A virus subtype H1, influenza A virus subtype H1 (2009), influenza A virus subtype H3, influenza B virus, RSV, hMPV, rhinovirus/enterovirus, adenovirus, parainfluenza viruses 1 to 4, coronavirus OC43, and coronavirus HKU1. xTAG RVP Fast was FDA cleared in July 2011 but only for the detection of 8 viruses and subtypes, including influenza A virus, influenza A virus subtype H1, influenza A virus subtype H3, influenza B virus, RSV, hMPV, rhinovirus, and adenovirus. Additionally, both assays are FDA cleared only for nasopharyngeal swabs, leaving other specimen types (sputum, bronchoscopic washes, lavages, etc.) to be validated by the individual laboratories.

FA RVP was more sensitive than xTAG RVP Fast, with a turnaround time of approximately 1 h for each specimen. The simplicity and the random-access characteristic make it an excellent choice in a hospital with low to medium volume and on-demand urgent service.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the Society of the Memorial Sloan-Kettering Cancer Center (to N.E.B.).

We acknowledge the virology staff for help with the collection of respiratory specimens and performance of the FA RVP assay.

Footnotes

Published ahead of print 18 April 2012

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