Skip to main content
PMC home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2011 Dec 28;72(3):278–281. doi: 10.1016/j.diagmicrobio.2011.11.005

Evaluation of xTAG Respiratory Viral Panel FAST and xTAG Human Parainfluenza Virus Analyte-Specific Reagents for detection of human parainfluenza viruses in respiratory specimens☆

Suresh B Selvaraju 1,2, Rangaraj Selvarangan 1,2,⁎
PMCID: PMC7172276  PMID: 22209513

Abstract

The multiplex xTAG® Respiratory Viral Panel FAST (RVP FAST) research-use-only assay and xTAG® Human Parainfluenza Virus Analyte-Specific Reagent (HPIV-ASR) assay were evaluated with 99 culture-confirmed human parainfluenza virus (HPIV)–positive and –negative specimens and found to have analytical sensitivities of 95.2% and 100% and specificities of 98.3% and 96.6%, respectively. Since the in vitro diagnostic (IVD) version of the RVP FAST assay does not include HPIVs, the HPIV-ASR assay can be tested in parallel with RVP FAST-IVD for optimal detection of HPIVs.

Keywords: Respiratory viruses, Human parainfluenza viruses, xTAG RVP FAST, xTAG Human Parainfluenza Virus ASR, Analyte-specific reagents

1. Introduction

Nucleic acid–based assays have been developed for accurate and sensitive detection of respiratory viruses with a shorter turnaround time when compared to traditional methods (Gunson et al., 2005, Heim et al., 2003, Mahony, 2008, Pabbaraju et al., 2007). Most importantly, multiplex polymerase chain reaction (PCR) tests have recently been introduced to detect several respiratory virus types and subtypes that include newly identified viruses such as human metapneumovirus, coronaviruses, human bocavirus, 2009 H1N1 virus, etc., in addition to the respiratory viruses routinely isolated in a clinical laboratory (Arens et al., 2010, Balada-Llasat et al., 2011, Brunstein and Thomas, 2006, Gharabaghi et al., 2011, Lee et al., 2007, Mahony et al., 2007, Nolte et al., 2007, Rand et al., 2011, Scheltinga et al., 2005). One of the multiplex PCR-based assays, xTAG respiratory viral panel assay (RVP-v1), was the first assay cleared by the Food and Drug Administration for clinical testing of respiratory viruses. The performance characteristics of this assay have been reported and widely accepted for excellent detection of respiratory viruses in clinical specimens (Balada-Llasat et al., 2011, Gharabaghi et al., 2011, Krunic et al., 2007, Mahony et al., 2007, Pabbaraju et al., 2008, Rand et al., 2011). Recently, a second generation of this assay called xTAG RVP FAST (RVP FAST) with a faster assay time was approved for in vitro diagnostic (IVD) use in the USA. This IVD version detects 8 respiratory viruses but does not include human parainfluenza viruses (HPIVs). As an alternate approach, the manufacturer has developed a new xTAG HPIV Analyte-Specific Reagent (HPIV-ASR) to detect all human parainfluenza virus types. HPIV-ASR can be performed in parallel with RVP FAST assay in the same 96-well plate, with same nucleic acid extracts, and tested under the same assay and analysis conditions. Here we report the performance of the research-use-only version of the RVP FAST and the HPIV-ASR assays in detecting HPIVs in respiratory specimens.

A total of 99 respiratory specimens (HPIV positive = 41 and HPIV negative = 58) that were tested for HPIV by either virology culture (n = 89) or RVP-v1 testing (n = 10) were selected for this study. HPIV positives include 15 HPIV-1, 2 HPIV-2, and 24 HPIV-3. The specimens submitted for RVP-v1 testing were culture confirmed for inclusion into this study.

The sensitivity of the RVP FAST and HPIV-ASR assays was determined by using human parainfluenza reference strains obtained from American Type Culture Collection (ATCC). The initial viral titer (50% tissue culture infective dose [TCID50]/0.2 mL) of HPIV reference strains was as follows: HPIV-1 = 1 × 103.75 (ATCC no. VR-94); HPIV-2 = 1 × 105.75 (ATCC no. VR-92); HPIV-3 = 1 × 107.5 (ATCC no. VR-93), and HPIV-4 = 1 × 103.5 (ATCC no. VR-1378). The HPIV strains were diluted 10-fold serially in universal transport medium and tested at different viral titer levels in triplicates as shown in Table 1 .

Table 1.

Limit of detection of xTAG RVP FAST and HPIV-ASR assays in detecting human parainfluenza virusesa

Viral target Viral titer dilutions as 1× TCID50/0.2 mL HPIV detection/typing
RVP FAST
 HPIV-ASR
Detection (MFI) Typing Detection (MFI) Resultb
HPIV-1 100.75 6168 HPIV-1 16,549 Positive
100.075 5699 HPIV-1 16,275 Positive
100.0075 3003 HPIV-1 16,453 Positive
100.00075 394 HPIV-1 9301 Positive
100.000075 50 Negative 871 Positive
100.0000075 62 Negative 123 Negative
100.00000075 69 Negative 94 Negative
HPIV-2 102.75 6624 HPIV-2 17,310 Positive
101.75 7048 HPIV-2 17,336 Positive
100.75 3960 HPIV-2 16,783 Positive
100.075 457 HPIV-2 12,067 Positive
100.0075 83 Negative 2346 Positive
100.00075 74 Negative 99 Negative
100.000075 65 Negative 94 Negative
HPIV-3 104.5 6035 HPIV-3 16,471 Positive
103.5 3293 HPIV-3 16,400 Positive
102.5 193 HPIV-3 7690 Positive
101.5 37 Negative 748 Positive
100.5 43 Negative 123 Negative
100.05 57 Negative 107 Negative
HPIV-4 100.05 2267 HPIV-4 10,352 Positive
100.005 67 Negative 1161 Positive
100.0005 62 Negative 133 Negative
100.00005 82 Negative 135 Negative
100.000005 51 Negative 190 Negative
100.0000005 47 Negative 108 Negative
Open in a new tab

The limit of detection (LoD) is indicated in italic type. RVP FAST = xTAG Respiratory Viral Panel Fast assay; HPIV-ASR = xTAG Human Parainfluenza Virus Analyte-Specific Reagents; MFI = median fluorescence intensity.

a

The reference HPIV strains were serially diluted 10-fold using universal transport medium for viruses and tested in triplicates.

b

HPIV-ASR detects HPIV 1–4 subtypes but does not differentiate them.

Total nucleic acids were extracted with MS2 internal control (MS2 added per manufacturer's instructions) using the NucliSENS easyMAG automated system (bioMérieux, Durham, NC, USA) and tested using RVP FAST assay (Luminex, Austin, TX, USA) according to the manufacturer's instructions in a 96-well plate format.

The HPIV-ASR (Luminex) assay was performed with the same nucleic acid extracts used for RVP FAST testing in the same 96-well plate along with the RVP FAST assay. A 20-μL reaction volume containing xTAG RNase-free water (2.3 μL), xTAG 5× OneStep Buffer (4.0 μL), xTAG dNTP mix (1.1 μL), PIV primer (0.8 μL), MS2 primer (0.2 μL), xTAG OneStep enzyme mix (1.6 μL), and template nucleic acid (10 μL) was setup. The PCR was performed with the RVP FAST reaction conditions. The MagPlex-TAG magnetic beads (Luminex) for PIVs and MS2 internal control are supplied separately and are mixed before performing bead hybridization. Twenty microliters of bead mix per reaction was prepared by mixing 1 μL each of MagPlex-TAG-025 (HPIV) and MagPlex-TAG-055 (MS2) beads with 18 μL of 1× xTAG hybridization buffer. A 75-μL reporter solution per reaction was prepared with 1 μL of SA-PE G75 conjugate and 74 μL of 1× xTAG reporter buffer. Bead hybridization was performed with the RVP FAST reaction conditions. Following incubation, the plate was then read using the Luminex 100 xPONENT 3.1 system (Luminex) and the raw data was analyzed per RVP FAST protocol.

The limit of detection (LoD) analysis with HPIV reference strains is shown in Table 1. For the RVP FAST assay, the LoD was 100.00075 TCID50/0.2 mL for HPIV-1, 100.075 for HPIV-2, 102.5 for HIPV-3, and 100.05 for HPIV-4. For the HPIV-ASR assay, the LoD was 100.000075 for HPIV-1, 100.0075 for HPIV-2, 101.5 for HPIV-3, and 100.005 for HPIV-4. In each case, the HPIV-ASR assay was capable of detecting HPIVs through 1 additional dilution compared to the RVP FAST assay.

The study results showed comparable performance of both RVP FAST and HPIV-ASR versus the gold standard culture method in detecting HPIV in clinical specimens (Table 2 ). RVP FAST and HPIV-ASR showed overall analytical sensitivities of 95.2% and 100% and specificities of 98.3% and 96.6%, respectively. The RVP FAST assay failed to detect 1 HPIV-3 positive and incorrectly typed 1 HPIV-1 positive as HPIV-3. Both of these discrepant specimens were tested by RVP-v1 and repeat tested by RVP FAST. The RVP-v1 assay detected both specimens accurately. The repeat RVP FAST testing detected HPIV-1 correctly, but failed to detect the HPIV-3–positive specimen. The inability of the RVP FAST assay to detect this HPIV-3–positive specimen could be attributed to the low sensitivity of this assay as the median fluorescence intensity (MFI) on RVP FAST assay was 169 for HPIV-3 compared to RVP-v1 MFI of 2726 and HPIV-ASR MFI of 8644. Earlier reports also suggested lower sensitivity of 33.3% (Gadsby et al., 2010) and 42.8% (Gharabaghi et al., 2011) by RVP FAST assay in detecting HPIV-3 type. Overall, our study results demonstrated that the RVP FAST assay produced lower MFI for all HPIV-3 positives as compared to the HPIV-ASR assay with mean MFI difference of 3419.6. In contrast, Pabbaraju et al. (2011) reported a sensitivity of 100% for HPIV-3; such differences in detection rates may be explained by regional/geographical strain differences. Interestingly, the same report showed a lower sensitivity of 63.6% for HPIV-2 compared to 100% in our study. However, more HPIV-2–positive samples should be analyzed to draw definitive conclusions as only 2 positive specimens were analyzed in our study. HPIV-1 was detected at the same sensitivity level of 93.3% as reported earlier (Pabbaraju et al., 2011).

Table 2.

Detection of human parainfluenza viruses by xTAG RVP FAST and HPIV-ASR assays in respiratory specimens

xTAG assays Viral target Detected/culture confirmed Percentage (95% confidence interval)
Sensitivity Specificity Positive predictive value Negative predictive value
RVP FAST Overall 39/41a,b 95.2a (82.6–99.2) 98.3b (89.7–99.9) 97.6 (85.6–99.9) 96.7 (87.5–99.4)
HPIV-1 14/15b 93.3b (66–99.7) 98.3b (89.7–99.9) 93.3 (66–99.7) 100 (92.3–100)
HPIV-2 2/2 100 (19.7–100) 100 (92.3–100) 100 (19.7–100) 100 (92.3–100)
HPIV-3 23/24 95.8a (76.9–99.8) 100 (92.3–100) 100 (82.2–100) 98.3 (89.7–99.9)
HPIV-ASR Overall 41/41 100 (89.3–100) 96.6c (87.0–99.4) 95.4 (82.9–99.2) 100 (92–100)
HPIV-1 15/15 100 (74.7–100) 100 (92–100) 100 (74.7–100) 100 (92–100)
HPIV-2 2/2 100 (19.8–100) 100 (92–100) 100 (19.8–100) 100 (92–100)
HPIV-3 24/24 100 (82.8–100) 100 (92–100) 100 (82.8–100) 100 (92–100)
Open in a new tab

The sensitivity and specificity of RVP FAST and HPIV-ASR assays were determined by comparing with culture as gold standard.

Both specimens were negative by discrepant analysis with RVP-v1 and repeat HPIV-ASR assays.

a

RVP FAST assay failed to detect 1 HPIV-3 culture positive.

b

RVP FAST assay typed 1 HPIV-1 type as HPIV-3 type initially. On discrepant analysis, RVP-v1 and RVP FAST assays typed this sample correctly as HPIV-1.

c

HPIV-ASR detected 2 culture-negative specimens as HPIV positives.

The HPIV-ASR accurately detected all HPIV types (100%; n = 41/41); however, 2 culture-negative specimens were initially detected as HPIV positives which reduced the specificity of this assay to 96.6%. These 2 specimens were confirmed as “false-positive results” since discrepant analysis result using both RVP-v1 assay and repeat HPIV-ASR was negative.

In summary, the RVP FAST assay and the HPIV-ASR performed well in comparison to culture in detecting HPIVs with our clinical specimens. Earlier studies using the RVP FAST research-use-only version reported comparatively lower sensitivity of this assay in detecting HPIVs (Gadsby et al., 2010, Gharabaghi et al., 2011, Pabbaraju et al., 2011). However, these studies were conducted in other regional/geographical regions, and in this study we achieved slightly higher sensitivity using the HPIV-ASR assay. As HPIVs are not included in the US IVD RVP FAST assay, clinical laboratories may choose to include HPIV-ASR assay in parallel with the US IVD RVP FAST assay in the same 96-well plate. The reverse transcription–PCR amplification and bead hybridization for both RVP FAST and HPIV-ASR can be performed with the same nucleic acid extract under the same reaction conditions. Reading and analyzing the assay results can also be performed at the same time by creating a multi-batch with the xPONENT software. Since separate extraction of nucleic acid is not necessary, there will not be a considerable difference in total time except for setting up of the PCR assay and preparing bead hybridization reactions. We estimate a total time/hands-on time of 5.5 h/1.75 h (extraction = 90 min/40 min; multiplex RT-PCR = 155 min/35 min; hybridization and detection = 35 min/15 min; and reading and analysis = 30 min/5 min), to process a batch size of 24–48 specimens. When compared with the xTAG respiratory viral panel assay (RVP-v1), the use of the RVP FAST assay will result in cost savings due to a reduced hands-on time of approximately 2 h, in addition to an improved turnaround time as a result of the shorter protocol.

Footnotes

☆

This study was funded by a research grant from Luminex Corporation.

References

  1. Arens M.Q., Buller R.S., Rankin A., Mason S., Whetsell A., Agapov E., Lee W.M., Storch G.A. Comparison of the Eragen Multi-Code Respiratory Virus Panel with conventional viral testing and real-time multiplex PCR assays for detection of respiratory viruses. J. Clin. Microbiol. 2010;48:2387–2395. doi: 10.1128/JCM.00220-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balada-Llasat J.M., LaRue H., Kelly C., Rigali L., Pancholi P. Evaluation of commercial ResPlex II v2.0, MultiCode-PLx, and xTAG respiratory viral panels for the diagnosis of respiratory viral infections in adults. J. Clin. Virol. 2011;50:42–45. doi: 10.1016/j.jcv.2010.09.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunstein J., Thomas E. Direct screening of clinical specimens for multiple respiratory pathogens using the Genaco Respiratory Panels 1 and 2. Diagn. Mol. Pathol. 2006;15:169–173. doi: 10.1097/01.pdm.0000210430.35340.53. [DOI] [PubMed] [Google Scholar]
  4. Gadsby N.J., Hardie A., Class E.C.J., Templeton K.E. Comparison of the Luminex Respiratory Viral Panel Fast assay with in-house real-time PCR for respiratory viral infection diagnosis. J. Clin. Microbiol. 2010;48:2213–2216. doi: 10.1128/JCM.02446-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gharabaghi F., Hawan A., Drews S.J., Richardson S.E. Evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children. Clin. Microbiol. Infect. 2011;17:1900–1906. doi: 10.1111/j.1469-0691.2011.03529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gunson R.N., Collins T.C., Carman W.F. Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reaction. J. Clin. Microbiol. 2005;33:341–344. doi: 10.1016/j.jcv.2004.11.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heim A., Ebnet C., Harste G., Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR. J. Med. Virol. 2003;70:228–239. doi: 10.1002/jmv.10382. [DOI] [PubMed] [Google Scholar]
  8. Krunic N., Yager T.D., Himsworth D., Merante F., Yaghoubian S., Janeczko R. xTAG RVP assay: analytical and clinical performance. J. Clin. Microbiol. 2007;40(S):S39–S46. doi: 10.1016/S1386-6532(07)70009-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lee W.M., Grindle K., Pappas T., Marshall D.J., Moser M.J., Beaty E.L., Shult P.A., Prudent J.R., Gern J.E. High-throughput, sensitive, and accurate multiplex PCR-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses. J. Clin. Microbiol. 2007;45:2626–2634. doi: 10.1128/JCM.02501-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mahony J.B. Detection of respiratory viruses by molecular methods. Clin. Microbiol. Rev. 2008;21:716–747. doi: 10.1128/CMR.00037-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mahony J.B., Chong S., Merante F., Yaghoubian S., Sinha T., Lisle C., Janeczko R. 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. J. Clin. Microbiol. 2007;45:2965–2970. doi: 10.1128/JCM.02436-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nolte F.S., Marshall D.J., Rasberry C., Schievelbein S., Banks G.G., Storch G.A., Arens M.Q., Buller R.S., Prudent J.R. MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses. J. Clin. Microbiol. 2007;45:2779–2786. doi: 10.1128/JCM.00669-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pabbaraju J., Wong S., Tokaryk K.L., Fonseca K., Drews S.J. Comparison of the Luminex xTAG respiratory viral panel with xTAG respiratory viral panel fast for diagnosis of respiratory virus infections. J. Clin. Microbiol. 2011;49:1738–1744. doi: 10.1128/JCM.02090-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pabbaraju K., Wong S., McMillan T., Lee B.E., Fox J.D. Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR. J. Clin. Virol. 2007;40:186–192. doi: 10.1016/j.jcv.2007.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pabbaraju K., Tokaryk K.L., Wong S., Fox J.D. Comparison of the Luminex xTAG respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections. J. Clin. Microbiol. 2008;46:3056–3062. doi: 10.1128/JCM.00878-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rand K.H., Rampersaud H., Houck H.J. A comparison of two multiplex methods for the detection of respiratory viruses: FilmArray RP and xTAG RVP. J. Clin. Microbiol. 2011;49:2449–2453. doi: 10.1128/JCM.02582-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Scheltinga S.A., Templeton K.E., Beersma M.F., Claas E.C.J. Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR. J. Clin. Virol. 2005;33:306–311. doi: 10.1016/j.jcv.2004.08.021. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Diagnostic Microbiology and Infectious Disease are provided here courtesy of Elsevier

RESOURCES