- Email: [email protected]
B & RSV kit (nucleic acid extraction–dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses
Comparison of the Simplexa TM Flu A/B & RSV kit (nucleic acid extractiondependant assay) and the Prodessa ProFlu + TM Assay for Detecting Influenza and Respiratory Syncytial Viruses Suresh B. Selvaraju, Adrienne V. Bambach, Amy L. Leber, Maria-Magdalena Patru, Anami Patel, Marilyn A. Menegus PII: DOI: Reference:
S0732-8893(13)00628-7 doi: 10.1016/j.diagmicrobio.2013.11.015 DMB 13477
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
24 June 2013 12 November 2013 18 November 2013
Please cite this article as: Selvaraju Suresh B., Bambach Adrienne V., Leber Amy L., Patru Maria-Magdalena, Patel Anami, Menegus Marilyn A., Comparison of the SimplexaTM Flu A/B & RSV kit (nucleic acid extraction-dependant assay) and the Prodessa ProFlu + TM Assay for Detecting Influenza and Respiratory Syncytial Viruses, Diagnostic Microbiology and Infectious Disease (2013), doi: 10.1016/j.diagmicrobio.2013.11.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extractiondependant assay) and the Prodessa ProFlu+™ Assay for Detecting Influenza and Respiratory Syncytial Viruses
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Suresh B. Selvaraju1‡, Adrienne V. Bambach1‡, Amy L. Leber2, Maria-Magdalena Patru1, Anami Patel3 and Marilyn A. Menegus1* University of Rochester Medical Center, Rochester, NY 14642, 2Nationwide Children's Hospital, Columbus, OH 43205, and 3LeBonheur Children’s Hospital, Memphis, TN 38103
Authors contributed equally
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______________ Corresponding author: Mailing address: Clinical Microbiology Laboratory, University of Rochester Medical Center, 601 Elmwood Avenue, Box 710, Rochester, NY 14642. Tel.: 585275-7735; E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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The relative performance of two widely used RT-PCR assays, the Focus diagnostics Simplexa™
3
Flu A/B & RSV kit (nucleic acid extraction-dependant assay) and the Prodessa Proflu+™ assay
4
was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab
5
specimens. Overall, the assays showed good agreement with positive and negative agreements of
6
100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B and 99.3% and 99.5% for
7
respiratory syncytial virus. The relative analytical sensitivity of the two assays was also similar.
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Key Words
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Simplexa, FOCUS nucleic acid extraction-dependant assay, Flu and RSV detection, Prodessa
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Proflu assay
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2
ACCEPTED MANUSCRIPT Influenza (Flu) and respiratory syncytial viruses (RSV) are the leading cause of severe
13
respiratory tract infections in young children, the elderly, pregnant women and persons with
14
chronic medical conditions (Falsey et al., 2005; Hall et al., 2009; CDC 2012). Rapid and accurate
15
methods for the diagnosis of respiratory viral infections are important for early antiviral
16
treatment, prophylaxis, and infection prevention. At present, the real-time PCR based molecular
17
methods are the preferred approach for the diagnosis offor diagnosing respiratory viral infections
18
due to their higher sensitivity and specificity compared to the insensitive rapid antigen tests, time
19
consuming virus culture and labor-intensive direct fluorescent antibody staining (DFA) (CDC,
20
2009). Focus diagnostics has developed a RT-PCR assay namely Simplexa™ Flu A/B & RSV
21
kit (Simplexa), which is a nucleic acid extraction-dependant assay that can process 96 samples
22
with its proprietary Universal disc on 3M Integrated cycler platform. The aim of this study was
23
to collect data for the FDA 510k submission of this Simplexa assay with the Prodessa ProFlu+™
24
(Proflu; Hologic, Bedford, MA) assay being used as one of the predicate devices along with the
25
culture and/or DFA traditional methods. However, here we report only the performance of this
26
Simplexa assay against the Prodessa assay in detecting Flu A, Flu B and RSV viruses in
27
nasopharyngeal swab specimens.
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A total of 735 nasopharyngeal swab specimens in viral transport medium were collected
29
at different sites of the USA (2 sites) and Australia (1 site). Of 735 specimens, 558 were
30
prospectively collected from the USA in the months of February and March 2010 and from
31
Australia in the months of July and August 2010. The remaining 177 specimens were
32
retrospectively collected from 2 USA sites during the 2008 – 2009 and early 2010 influenza and
33
RSV seasons from patients with signs and symptoms of viral respiratory tract infection. For
34
prospective specimen collection, male and female subjects from the general population 3
ACCEPTED MANUSCRIPT exhibiting clinical symptoms of an upper respiratory infection were eligible for inclusion in this
36
study. The clinical specimens (retrospective and prospective) were collected from patients in the
37
age range of 4 months to 91 years old and from roughly equal numbers of males (387) and
38
females (348). Both prospectively and retrospectively collected specimens were shipped on dry
39
ice to three different clinical trial sites for testing.
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T
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The relative analytical sensitivity of the Simplexa and the Proflu assays for Flu A, B and
41
RSV was investigated at one site. The experiment was performed with one clinical viral isolate
42
each for Flu A H1 seasonal, 2009 pandemic Flu A H1, Flu A H3, Flu B, RSV A and RSV B.
43
Viral isolates of Flu were typed by World Health Organization (WHO) Influenza Reagent Kit,
44
which was supplied by CDC as part of WHO Collaborating Center for Surveillance,
45
Epidemiology and Control of Influenza program. The RSV A and RSV B were obtained from
46
Dr. Caroline Hall, University of Rochester. The 50% tissue culture infectious doses (TCID50/ml)
47
of the test strains were determined at Strong Memorial Hospital virology laboratory as follows.
48
Flu and RSV viral strains were grown in primary Rhesus Monkey Kidney (RhMK) and Human
49
Epidermoid Cancer (HEp-2) cells, respectively. The lysates of the infected cell cultures were
50
then diluted 5-fold in M5 viral transport medium (VTM; Remel, Lenexa, KS) and titrated in
51
duplicate in RhMK cells for Flu and HEp-2 cells for RSV using Spearman-Karber method
52
(Hamilton et al., 1977). All the test strains were serially diluted 10-fold with M5 VTM and tested
53
with the Simplexa and Proflu assays.
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With the aim of getting FDA clearance for MagNA Pure LC 2.0 (Roche, Indianapolis,
55
IN) and NucliSENS® easyMAG™ (bioMérieux, Durham, NC) automated nucleic acid extraction
56
systems for clinical testing, both extraction systems were included in the clinical trial testing.
57
Two clinical trial sites tested 435/735 specimens using MagNA Pure system and another clinical 4
ACCEPTED MANUSCRIPT trial site tested the remaining 300/735 specimens using the easyMAG system. Using both nucleic
59
acid extraction systems, the total nucleic acid was eluted in 50 μl volume from 200 μl of
60
specimen and 5 μl of the nucleic acid was used as template to perform the Simplexa assay using
61
96-well universal disc and 3M Integrated Cycler according to the manufacturer’s instructions.
62
All Proflu assay testing was performed at one USA site where the nucleic acid was extracted
63
from 200 μl of specimen using the MagNA Pure Compact System (Roche) with an elution
64
volume of 50 μl and 5 μl of the extract was used to perform the assay on the SmartCycler
65
(Cepheid, Sunnyvale, CA) according to the package insert. Each assay run included appropriate
66
positive, negative and internal controls when testing clinical specimens.
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Statistical analyses were performed on qualitative results of the assays per study protocol.
68
The sample size used in this clinical study was calculated using the binomial distribution with
69
the lower confidence bound as the acceptance criterion. Wilson’s score interval method and
70
linear mixed effect model were used to calculate confidence interval of binomial proportion and
71
variance component, respectively.
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67
72
The analytical sensitivity study results showed an excellent agreement between the
73
Simplexa and the Proflu assays. The Simplexa assay showed 1-log increased sensitivity for Flu
74
A-H1 seasonal, Flu A-H1 pdm09 and RSV B viruses compared to the Proflu assay (Table 1). All
75
other test strains showed similar level of detection, which correlate with the comparable clinical
76
performance of these two assays.
77
Overall, the Simplexa assay showed remarkable agreement with the Proflu assay in
78
detecting Flu A, Flu B and RSV in clinical specimens. Of the 735 specimens received, 730 were
79
considered for analysis and the remaining five specimens were excluded for various reasons 5
ACCEPTED MANUSCRIPT including invalid or unresolved results by any RT-PCR assay. The overall (prospective and
81
retrospective) positive / negative percent agreement of the Simplexa assay with the Proflu was
82
determined to be 100% / 99.7% for Flu A, 98.1% / 99.9% for Flu B and 99.3% / 99.5% for RSV
83
(Table 2). Hindiyeh and co-workers showed similar good performance of the Simplexa assay in
84
detecting all three viral targets compared to their laboratory developed assay (LDT) (Hindiyeh et
85
al., 2013). Although that study results showed lesser sensitivity compared to our results in
86
detecting Flu A and RSV, it is understandable to have lower sensitivity with extraction-free
87
format of their assay. The Simplexa assay showed a trend of yielding lower Ct values compared
88
to the Proflu assay. The mean Ct differences between the assays were determined to be 3.4
89
(range=0.2-5.8) for Flu A, 0.8 (range=0-4.1) for Flu B and 1.86 (range=0-6.8) for RSV. This
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could be attributed to many different factors, including, but not limited to, differences in assay
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chemistry, nucleic acid extraction methods, assay thresholds and assay sensitivity.
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A recent publication by Alby and co-workers reported a lower sensitivity of the Simplexa
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assay for Flu A and Flu B viruses (Alby et al., 2013). In contrast, our study showed an excellent
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performance of the Simplexa assay in detecting Flu and RSV viruses with more number of
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specimens collected from diverse patient population from different parts of the USA and
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Australia. The 20 specimens not detected by the Simplexa assay yielded the mean Ct value
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around 35 with their LDT. These higher Ct values indicate that both their LDT and the Verigene
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RV+ assays may run for extended cycles compared to the Simplexa assay. Hence, the assay
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format of the Simplexa assay might be the reason for not detecting those low viral burden
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specimens in their study. Further, the differences between our results and the previous study
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results might be due to differences in performance between the Proflu and the Verigene RV+
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assays. In this study, we used more standardized and well characterized Proflu assay as the
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ACCEPTED MANUSCRIPT comparator. Several earlier studies have used the Proflu assay as a reliable comparator in
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evaluating the assay performance characteristics in detecting Flu and RSV viruses (Selvaraju and
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Selvarangan, 2010; Loeffelholz et al., 2011; Novak-Weekley et al., 2012; Tang et al., 2013).
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No significant differences were observed in the assay results due to two different nucleic
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acid extraction systems used in this study to evaluate the Simplexa assay. All specimens
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extracted with MagNA pure LC 2.0 system (n=435/435) yielded concordant results between the
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Simplexa and Proflu assays while 2 specimens out of 300 extracted with easyMAG system
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yielded invalid results with the Simplexa assay (data not shown).
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In summary, the Simplexa assay showed equivalent performance with the Proflu assay
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for detection of Flu A, Flu B and RSV with additional advantages of a shorter run time (~1 hour)
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compared to the Proflu assay and the ability to process up to 96 specimens (including controls)
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per run. Hence, the FDA cleared SimplexaTM Flu A/B & RSV assay is a simple and reliable
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assay for sensitive and accurate detection of Flu and RSV viral infections in all laboratory
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settings.
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Acknowledgements
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The authors acknowledge Dr. Caroline Hall, MD, University of Rochester Medical Center,
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Rochester, NY for providing RSV A and B viruses. The authors also acknowledge Christine
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Mayer, University of Rochester Medical Center; Carol Cummins and Tim Peterson, Nationwide
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Children's Hospital; Noel Lenny and Joseph Davis, LeBonheur Children’s Hospital for their
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assistance in this study.
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ACCEPTED MANUSCRIPT References
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1. Alby K, Popowitch EB, Miller MB (2013) Comparative evaluation of the Nanosphere Verigene RV+ assay and the Simplexa Flu A/B & RSV kit for detection of influenza and respiratory syncytial viruses. J Clin Microbiol 51:352–353.
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2. Centers for Disease Control and Prevention (2009) Evaluation of rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus-US, 2009. Morbid Mortal Weekly Rep 58:826–829.
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3. Centers for Disease Control and Prevention (2012) Seasonal Influenza – Questions & Answers. http://www.cdc.gov/flu/about/qa/disease.htm. Accessed July 4th, 2012.
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4. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE (2005) Respiratory Syncytial Virus Infections in Elderly and High-risk Adults. N Engl J Med 352:1749–1759.
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5. Hall CB, Weinberg GA, Iwane MK, Blumkin AK, Edwards KM, Staat MA, Auinger P, Griffin MR, Poehling KA, Erdman D, Grijalva CG, Zhu Y, Szilagyi P (2009) The burden of respiratory syncytial virus infection in young children. N Engl J Med 360:588–598.
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6. Hamilton MA, Russo RC, Thurston RV (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in bioassays. Environ Sci Technol 11:714–719.
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7. Hindiyeh M, Kolet L, Meningher T, Weil M, Mendelson E, Mandelboim M (2013) Evaluation of Simplexa Flu A/B & RSV for direct detection of influenza viruses (A and B) and respiratory syncytial virus in patient clinical samples. J Clin Microbiol 51:2421-2424.
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8. Loeffelholz MJ, Pong DL, Pyles RB, Xiong Y, Miller AL, Bufton KK, Chonmaitree T (2011) Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens. J Clin Microbiol 49:4083–4088.
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9. Novak-Weekley SM, Marlowe EM, Poulter M, Dwyer D, Speers D, Rawlinson W, Baleriola C, Robinson CC (2012) Evaluation of the Cepheid Xpert Flu assay for rapid identification and differentiation of influenza A, influenza A 2009 H1N1, and influenza B viruses. J Clin Microbiol 50:1704–1710. 10. Selvaraju SB, Selvarangan R (2010) Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses. J Clin Microbiol 48:3870–3875. 11. Tang YW, Lowery KS, Valsamakis A, Schaefer VC, Chappell JD, White-Abell J, Quinn CD, Li H, Washington CA, Cromwell J, Giamanco CM, Forman M, Holden J, Rothman RE, Parker ML, Ortenberg EV, Zhang L, Lin YL, Gaydos CA (2013) Clinical accuracy of a PLEX-ID Flu device for simultaneous detection and identification of influenza viruses A and B. J Clin Microbiol 51:40–45.
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ACCEPTED MANUSCRIPT TABLE 1. Relative sensitivity of Simplexa and Proflu assays in detecting Flu A, B and RSV Viral target detection
28.4 32.1 35.0 39.4 0
32.3 34.5 37.7 0 0
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Proflu Ct
31.2 34.0 37.1 0 0
30.4 33.3 36.9 0 0
32.2 35.0 0 0 0
25.3 28.8 31.8 36.7 0
26.3 30.0 32.9 36.9 0
27.7 31.3 37.9 0 0
31.3 34.1 37.7 0 0
27.4 31.5 34.8 0 0
31.2 34.2 0 0 0
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30.0 33.1 36.8 0 0
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Flu A-H1 Seasonal 10-4 (2.59) 10-5 (1.59) 10-6 (0.59) 10-7 (0.059) 10-8 (0.0059) Flu A-H3 10-4 (2.24) 10-5 (1.24 ) 10-6 (0.24 ) 10-7 (0.024) 10-8 (0.0024) Flu A-H1 pdm09 10-4 (1.89) 10-5 (0.89) 10-6 (0.089) 10-7 (0.0089) 10-8 (0.00089) Flu B 10-4 (3.64) 10-5 (2.64) 10-6 (1.64) 10-7 (0.64) 10-8 (0.064) RSV A 10-4 (2.01) 10-5 (1.01) 10-6 (0.01) 10-7 (0.001) 10-8 (0.0001) RSV B 10-4 (1.08) 10-5 (0.08) 10-6 (0.008) 10-7 (0.0008) 10-8 (0.00008)
Simplexa Ct
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Viral stock dilutions (1x10^TCID50/ml)
Simplexa: Simplexa™ Flu A/B & RSV Assay Proflu: Prodessa ProFlu+™ assay Ct: Cycle threshold TCID50: 50% tissue culture infectious dose 9
ACCEPTED MANUSCRIPT
Target detection
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TABLE 2. Agreement between Simplexa and Proflu in detecting Flu A, B and RSV in clinical specimens*
Positive percent Simplexa – / Simplexa – / agreement ; a / (a + c) Proflu – Proflu + (95% CI) (c) (d)
Flu A
104
2
0
Flu B
51
1
1
RSV
132
3
1
Negative percent agreement ; d / (b + d) (95% CI)
624
100% ; 104/104 (95.6% - 100%)
99.7% ; 624/626 (98.7% - 99.9%)
677
98.1% ; 51/52 (88.4% - 99.9%)
99.9% ; 677/678 (99.0% - 100%)
594
99.3% ; 132/133 (95.3% - 99.9%)
99.5% ; 594/597 (98.4% - 99.9%)
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Analyte
Simplexa + / Simplexa + / Proflu + Proflu – (a) (b)
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* Both prospectively and retrospectively collected specimens were included for analysis Simplexa: Simplexa™ Flu A/B & RSV Assay Proflu: Prodessa ProFlu+™ assay CI: Confidence interval
10
S0732-8893(13)00628-7 doi: 10.1016/j.diagmicrobio.2013.11.015 DMB 13477
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
24 June 2013 12 November 2013 18 November 2013
Please cite this article as: Selvaraju Suresh B., Bambach Adrienne V., Leber Amy L., Patru Maria-Magdalena, Patel Anami, Menegus Marilyn A., Comparison of the SimplexaTM Flu A/B & RSV kit (nucleic acid extraction-dependant assay) and the Prodessa ProFlu + TM Assay for Detecting Influenza and Respiratory Syncytial Viruses, Diagnostic Microbiology and Infectious Disease (2013), doi: 10.1016/j.diagmicrobio.2013.11.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extractiondependant assay) and the Prodessa ProFlu+™ Assay for Detecting Influenza and Respiratory Syncytial Viruses
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Suresh B. Selvaraju1‡, Adrienne V. Bambach1‡, Amy L. Leber2, Maria-Magdalena Patru1, Anami Patel3 and Marilyn A. Menegus1* University of Rochester Medical Center, Rochester, NY 14642, 2Nationwide Children's Hospital, Columbus, OH 43205, and 3LeBonheur Children’s Hospital, Memphis, TN 38103
Authors contributed equally
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______________ Corresponding author: Mailing address: Clinical Microbiology Laboratory, University of Rochester Medical Center, 601 Elmwood Avenue, Box 710, Rochester, NY 14642. Tel.: 585275-7735; E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract
2
The relative performance of two widely used RT-PCR assays, the Focus diagnostics Simplexa™
3
Flu A/B & RSV kit (nucleic acid extraction-dependant assay) and the Prodessa Proflu+™ assay
4
was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab
5
specimens. Overall, the assays showed good agreement with positive and negative agreements of
6
100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B and 99.3% and 99.5% for
7
respiratory syncytial virus. The relative analytical sensitivity of the two assays was also similar.
NU
SC
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T
1
Key Words
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Simplexa, FOCUS nucleic acid extraction-dependant assay, Flu and RSV detection, Prodessa
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Proflu assay
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2
ACCEPTED MANUSCRIPT Influenza (Flu) and respiratory syncytial viruses (RSV) are the leading cause of severe
13
respiratory tract infections in young children, the elderly, pregnant women and persons with
14
chronic medical conditions (Falsey et al., 2005; Hall et al., 2009; CDC 2012). Rapid and accurate
15
methods for the diagnosis of respiratory viral infections are important for early antiviral
16
treatment, prophylaxis, and infection prevention. At present, the real-time PCR based molecular
17
methods are the preferred approach for the diagnosis offor diagnosing respiratory viral infections
18
due to their higher sensitivity and specificity compared to the insensitive rapid antigen tests, time
19
consuming virus culture and labor-intensive direct fluorescent antibody staining (DFA) (CDC,
20
2009). Focus diagnostics has developed a RT-PCR assay namely Simplexa™ Flu A/B & RSV
21
kit (Simplexa), which is a nucleic acid extraction-dependant assay that can process 96 samples
22
with its proprietary Universal disc on 3M Integrated cycler platform. The aim of this study was
23
to collect data for the FDA 510k submission of this Simplexa assay with the Prodessa ProFlu+™
24
(Proflu; Hologic, Bedford, MA) assay being used as one of the predicate devices along with the
25
culture and/or DFA traditional methods. However, here we report only the performance of this
26
Simplexa assay against the Prodessa assay in detecting Flu A, Flu B and RSV viruses in
27
nasopharyngeal swab specimens.
AC
CE
PT
ED
MA
NU
SC
RI P
T
12
28
A total of 735 nasopharyngeal swab specimens in viral transport medium were collected
29
at different sites of the USA (2 sites) and Australia (1 site). Of 735 specimens, 558 were
30
prospectively collected from the USA in the months of February and March 2010 and from
31
Australia in the months of July and August 2010. The remaining 177 specimens were
32
retrospectively collected from 2 USA sites during the 2008 – 2009 and early 2010 influenza and
33
RSV seasons from patients with signs and symptoms of viral respiratory tract infection. For
34
prospective specimen collection, male and female subjects from the general population 3
ACCEPTED MANUSCRIPT exhibiting clinical symptoms of an upper respiratory infection were eligible for inclusion in this
36
study. The clinical specimens (retrospective and prospective) were collected from patients in the
37
age range of 4 months to 91 years old and from roughly equal numbers of males (387) and
38
females (348). Both prospectively and retrospectively collected specimens were shipped on dry
39
ice to three different clinical trial sites for testing.
SC
RI P
T
35
The relative analytical sensitivity of the Simplexa and the Proflu assays for Flu A, B and
41
RSV was investigated at one site. The experiment was performed with one clinical viral isolate
42
each for Flu A H1 seasonal, 2009 pandemic Flu A H1, Flu A H3, Flu B, RSV A and RSV B.
43
Viral isolates of Flu were typed by World Health Organization (WHO) Influenza Reagent Kit,
44
which was supplied by CDC as part of WHO Collaborating Center for Surveillance,
45
Epidemiology and Control of Influenza program. The RSV A and RSV B were obtained from
46
Dr. Caroline Hall, University of Rochester. The 50% tissue culture infectious doses (TCID50/ml)
47
of the test strains were determined at Strong Memorial Hospital virology laboratory as follows.
48
Flu and RSV viral strains were grown in primary Rhesus Monkey Kidney (RhMK) and Human
49
Epidermoid Cancer (HEp-2) cells, respectively. The lysates of the infected cell cultures were
50
then diluted 5-fold in M5 viral transport medium (VTM; Remel, Lenexa, KS) and titrated in
51
duplicate in RhMK cells for Flu and HEp-2 cells for RSV using Spearman-Karber method
52
(Hamilton et al., 1977). All the test strains were serially diluted 10-fold with M5 VTM and tested
53
with the Simplexa and Proflu assays.
AC
CE
PT
ED
MA
NU
40
54
With the aim of getting FDA clearance for MagNA Pure LC 2.0 (Roche, Indianapolis,
55
IN) and NucliSENS® easyMAG™ (bioMérieux, Durham, NC) automated nucleic acid extraction
56
systems for clinical testing, both extraction systems were included in the clinical trial testing.
57
Two clinical trial sites tested 435/735 specimens using MagNA Pure system and another clinical 4
ACCEPTED MANUSCRIPT trial site tested the remaining 300/735 specimens using the easyMAG system. Using both nucleic
59
acid extraction systems, the total nucleic acid was eluted in 50 μl volume from 200 μl of
60
specimen and 5 μl of the nucleic acid was used as template to perform the Simplexa assay using
61
96-well universal disc and 3M Integrated Cycler according to the manufacturer’s instructions.
62
All Proflu assay testing was performed at one USA site where the nucleic acid was extracted
63
from 200 μl of specimen using the MagNA Pure Compact System (Roche) with an elution
64
volume of 50 μl and 5 μl of the extract was used to perform the assay on the SmartCycler
65
(Cepheid, Sunnyvale, CA) according to the package insert. Each assay run included appropriate
66
positive, negative and internal controls when testing clinical specimens.
MA
NU
SC
RI P
T
58
Statistical analyses were performed on qualitative results of the assays per study protocol.
68
The sample size used in this clinical study was calculated using the binomial distribution with
69
the lower confidence bound as the acceptance criterion. Wilson’s score interval method and
70
linear mixed effect model were used to calculate confidence interval of binomial proportion and
71
variance component, respectively.
AC
CE
PT
ED
67
72
The analytical sensitivity study results showed an excellent agreement between the
73
Simplexa and the Proflu assays. The Simplexa assay showed 1-log increased sensitivity for Flu
74
A-H1 seasonal, Flu A-H1 pdm09 and RSV B viruses compared to the Proflu assay (Table 1). All
75
other test strains showed similar level of detection, which correlate with the comparable clinical
76
performance of these two assays.
77
Overall, the Simplexa assay showed remarkable agreement with the Proflu assay in
78
detecting Flu A, Flu B and RSV in clinical specimens. Of the 735 specimens received, 730 were
79
considered for analysis and the remaining five specimens were excluded for various reasons 5
ACCEPTED MANUSCRIPT including invalid or unresolved results by any RT-PCR assay. The overall (prospective and
81
retrospective) positive / negative percent agreement of the Simplexa assay with the Proflu was
82
determined to be 100% / 99.7% for Flu A, 98.1% / 99.9% for Flu B and 99.3% / 99.5% for RSV
83
(Table 2). Hindiyeh and co-workers showed similar good performance of the Simplexa assay in
84
detecting all three viral targets compared to their laboratory developed assay (LDT) (Hindiyeh et
85
al., 2013). Although that study results showed lesser sensitivity compared to our results in
86
detecting Flu A and RSV, it is understandable to have lower sensitivity with extraction-free
87
format of their assay. The Simplexa assay showed a trend of yielding lower Ct values compared
88
to the Proflu assay. The mean Ct differences between the assays were determined to be 3.4
89
(range=0.2-5.8) for Flu A, 0.8 (range=0-4.1) for Flu B and 1.86 (range=0-6.8) for RSV. This
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could be attributed to many different factors, including, but not limited to, differences in assay
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chemistry, nucleic acid extraction methods, assay thresholds and assay sensitivity.
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A recent publication by Alby and co-workers reported a lower sensitivity of the Simplexa
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assay for Flu A and Flu B viruses (Alby et al., 2013). In contrast, our study showed an excellent
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performance of the Simplexa assay in detecting Flu and RSV viruses with more number of
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specimens collected from diverse patient population from different parts of the USA and
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Australia. The 20 specimens not detected by the Simplexa assay yielded the mean Ct value
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around 35 with their LDT. These higher Ct values indicate that both their LDT and the Verigene
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RV+ assays may run for extended cycles compared to the Simplexa assay. Hence, the assay
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format of the Simplexa assay might be the reason for not detecting those low viral burden
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specimens in their study. Further, the differences between our results and the previous study
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results might be due to differences in performance between the Proflu and the Verigene RV+
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assays. In this study, we used more standardized and well characterized Proflu assay as the
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evaluating the assay performance characteristics in detecting Flu and RSV viruses (Selvaraju and
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Selvarangan, 2010; Loeffelholz et al., 2011; Novak-Weekley et al., 2012; Tang et al., 2013).
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No significant differences were observed in the assay results due to two different nucleic
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acid extraction systems used in this study to evaluate the Simplexa assay. All specimens
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extracted with MagNA pure LC 2.0 system (n=435/435) yielded concordant results between the
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Simplexa and Proflu assays while 2 specimens out of 300 extracted with easyMAG system
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yielded invalid results with the Simplexa assay (data not shown).
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In summary, the Simplexa assay showed equivalent performance with the Proflu assay
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for detection of Flu A, Flu B and RSV with additional advantages of a shorter run time (~1 hour)
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compared to the Proflu assay and the ability to process up to 96 specimens (including controls)
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per run. Hence, the FDA cleared SimplexaTM Flu A/B & RSV assay is a simple and reliable
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assay for sensitive and accurate detection of Flu and RSV viral infections in all laboratory
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settings.
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Acknowledgements
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The authors acknowledge Dr. Caroline Hall, MD, University of Rochester Medical Center,
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Rochester, NY for providing RSV A and B viruses. The authors also acknowledge Christine
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Mayer, University of Rochester Medical Center; Carol Cummins and Tim Peterson, Nationwide
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Children's Hospital; Noel Lenny and Joseph Davis, LeBonheur Children’s Hospital for their
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assistance in this study.
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ACCEPTED MANUSCRIPT References
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1. Alby K, Popowitch EB, Miller MB (2013) Comparative evaluation of the Nanosphere Verigene RV+ assay and the Simplexa Flu A/B & RSV kit for detection of influenza and respiratory syncytial viruses. J Clin Microbiol 51:352–353.
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2. Centers for Disease Control and Prevention (2009) Evaluation of rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus-US, 2009. Morbid Mortal Weekly Rep 58:826–829.
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3. Centers for Disease Control and Prevention (2012) Seasonal Influenza – Questions & Answers. http://www.cdc.gov/flu/about/qa/disease.htm. Accessed July 4th, 2012.
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4. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE (2005) Respiratory Syncytial Virus Infections in Elderly and High-risk Adults. N Engl J Med 352:1749–1759.
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5. Hall CB, Weinberg GA, Iwane MK, Blumkin AK, Edwards KM, Staat MA, Auinger P, Griffin MR, Poehling KA, Erdman D, Grijalva CG, Zhu Y, Szilagyi P (2009) The burden of respiratory syncytial virus infection in young children. N Engl J Med 360:588–598.
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6. Hamilton MA, Russo RC, Thurston RV (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in bioassays. Environ Sci Technol 11:714–719.
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7. Hindiyeh M, Kolet L, Meningher T, Weil M, Mendelson E, Mandelboim M (2013) Evaluation of Simplexa Flu A/B & RSV for direct detection of influenza viruses (A and B) and respiratory syncytial virus in patient clinical samples. J Clin Microbiol 51:2421-2424.
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8. Loeffelholz MJ, Pong DL, Pyles RB, Xiong Y, Miller AL, Bufton KK, Chonmaitree T (2011) Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens. J Clin Microbiol 49:4083–4088.
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9. Novak-Weekley SM, Marlowe EM, Poulter M, Dwyer D, Speers D, Rawlinson W, Baleriola C, Robinson CC (2012) Evaluation of the Cepheid Xpert Flu assay for rapid identification and differentiation of influenza A, influenza A 2009 H1N1, and influenza B viruses. J Clin Microbiol 50:1704–1710. 10. Selvaraju SB, Selvarangan R (2010) Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses. J Clin Microbiol 48:3870–3875. 11. Tang YW, Lowery KS, Valsamakis A, Schaefer VC, Chappell JD, White-Abell J, Quinn CD, Li H, Washington CA, Cromwell J, Giamanco CM, Forman M, Holden J, Rothman RE, Parker ML, Ortenberg EV, Zhang L, Lin YL, Gaydos CA (2013) Clinical accuracy of a PLEX-ID Flu device for simultaneous detection and identification of influenza viruses A and B. J Clin Microbiol 51:40–45.
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ACCEPTED MANUSCRIPT TABLE 1. Relative sensitivity of Simplexa and Proflu assays in detecting Flu A, B and RSV Viral target detection
28.4 32.1 35.0 39.4 0
32.3 34.5 37.7 0 0
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Proflu Ct
31.2 34.0 37.1 0 0
30.4 33.3 36.9 0 0
32.2 35.0 0 0 0
25.3 28.8 31.8 36.7 0
26.3 30.0 32.9 36.9 0
27.7 31.3 37.9 0 0
31.3 34.1 37.7 0 0
27.4 31.5 34.8 0 0
31.2 34.2 0 0 0
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30.0 33.1 36.8 0 0
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Flu A-H1 Seasonal 10-4 (2.59) 10-5 (1.59) 10-6 (0.59) 10-7 (0.059) 10-8 (0.0059) Flu A-H3 10-4 (2.24) 10-5 (1.24 ) 10-6 (0.24 ) 10-7 (0.024) 10-8 (0.0024) Flu A-H1 pdm09 10-4 (1.89) 10-5 (0.89) 10-6 (0.089) 10-7 (0.0089) 10-8 (0.00089) Flu B 10-4 (3.64) 10-5 (2.64) 10-6 (1.64) 10-7 (0.64) 10-8 (0.064) RSV A 10-4 (2.01) 10-5 (1.01) 10-6 (0.01) 10-7 (0.001) 10-8 (0.0001) RSV B 10-4 (1.08) 10-5 (0.08) 10-6 (0.008) 10-7 (0.0008) 10-8 (0.00008)
Simplexa Ct
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Viral stock dilutions (1x10^TCID50/ml)
Simplexa: Simplexa™ Flu A/B & RSV Assay Proflu: Prodessa ProFlu+™ assay Ct: Cycle threshold TCID50: 50% tissue culture infectious dose 9
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Target detection
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TABLE 2. Agreement between Simplexa and Proflu in detecting Flu A, B and RSV in clinical specimens*
Positive percent Simplexa – / Simplexa – / agreement ; a / (a + c) Proflu – Proflu + (95% CI) (c) (d)
Flu A
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2
0
Flu B
51
1
1
RSV
132
3
1
Negative percent agreement ; d / (b + d) (95% CI)
624
100% ; 104/104 (95.6% - 100%)
99.7% ; 624/626 (98.7% - 99.9%)
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98.1% ; 51/52 (88.4% - 99.9%)
99.9% ; 677/678 (99.0% - 100%)
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99.3% ; 132/133 (95.3% - 99.9%)
99.5% ; 594/597 (98.4% - 99.9%)
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Simplexa + / Simplexa + / Proflu + Proflu – (a) (b)
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* Both prospectively and retrospectively collected specimens were included for analysis Simplexa: Simplexa™ Flu A/B & RSV Assay Proflu: Prodessa ProFlu+™ assay CI: Confidence interval
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