Diagnostic Microbiology and Infectious Disease 78 (2014) 360–362

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Virology

Performance of the Directigen EZ Flu A+B rapid influenza diagnostic test to detect pandemic influenza A/H1N1 2009 Bobby L. Boyanton Jr. a, c,⁎, Amro Almradi a, Tejal Mehta b, Barbara Robinson-Dunn a, c a b c

Department of Clinical Pathology, Beaumont Health System, Royal Oak, MI Department of Infectious Diseases, Beaumont Health System, Royal Oak, MI Oakland University William Beaumont School of Medicine, Rochester, MI

a r t i c l e

i n f o

Article history: Received 19 May 2013 Received in revised form 30 September 2013 Accepted 6 October 2013 Available online 14 October 2013

a b s t r a c t The Directigen EZ Flu A+B rapid influenza diagnostic test, as compared to real-time reverse transcriptase polymerase chain reaction, demonstrated suboptimal performance to detect pandemic influenza A/H1N1 2009. Age- and viral load–stratified test sensitivity ranged from 33.3 to 84.6% and 0 to 100%, respectively. © 2014 Elsevier Inc. All rights reserved.

Keywords: Influenza A H1N1 2009 Swine flu Real-time PCR Directigen EZ Flu A+B Rapid influenza diagnostic test Diagnostic performance

Pandemic influenza A/H1N1 2009 first appeared in the United States in April 2009 (Dawoo et al., 2009). Shortly thereafter, the U.S. Centers for Disease Control and Prevention (CDC) released concerning information about the ability of rapid influenza diagnostic tests (RIDTs) to detect this virus (CDC, 2009b). Specifically, peer-reviewed literature pertaining to the performance of the Directigen EZ Flu A+B RIDT (Directigen-EZ; Becton Dickinson, Franklin Lakes, NJ, USA) to detect this virus is limited, despite being a commonly used RIDT (Chartrand et al., 2012). Reported sensitivity and specificity ranges for Directigen-EZ are 20.6–74.7% and 96.5–100%, respectively, when using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (qPCR) or the xTAG® Respiratory Viral Panel v1 as the gold standard (Luminex-RVP, Luminex Corp., Austin, TX, USA) (Al Johani et al., 2011; CDC, 2009b; Cheng et al., 2011; Herzum and Lutz, 2010; Karre et al., 2010; Vasoo et al., 2009; Welch and Ginocchio, 2010). Pre-analytical variables influencing RIDT performance include specimen collection adequacy, patient age, duration of illness, viral load, and the dilutional impact of viral transport media (CDC, 2011, 2012; Chartrand et al., 2012; Doller et al., 1992; Kaiser and Briones, 1999; Welch and Ginocchio, 2010). Due to the wide sensitivity range reported in the literature for the Directigen-EZ, we set out to characterize the impact of patient age and viral load on the performance of this RIDT to detect influenza A/H1N1 2009, under tightly controlled pre-analytical conditions.

⁎ Corresponding author. Tel.: +1-248-551-0130; fax: +1-248-551-0557. E-mail address: [email protected] (B.L. Boyanton). 0732-8893/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.10.009

This study was approved by the Human Investigative Committee (HIC) of Beaumont Health System. From October 2009 to January 2010 (second wave of influenza A/H1N1 2009 in Southeast Michigan), a single nasopharyngeal swab (NPS) specimen from each patient (n = 748, age 2 mo to 96 y) with influenza-like illness was obtained, placed into 3.0 mL of M4-RT transport medium (Remel, Lenexa, KS, USA) and tested by Directigen-EZ within 12 hours of collection. Total nucleic acid was purified from 200 μL of M4-RT on the NucliSENS easyMag (bioMerieux, Durham, NC, USA) using the Food and Drug Administration (FDA)–approved LuminexRVP extraction protocol, refrigerated, and tested within 24 hours using an in-house validated CDC real-time RT-PCR protocol for influenza A/H1N1 2009 (CDC-qPCR) (CDC, 2009a). Testing was performed on an ABI 7500 Fast (Life Technologies) and interpreted as described (CDC, 2009a). In brief, the protocol includes a panel of 4 oligonucleotide primer and dual-labeled hydrolysis probe sets. The InfA, swInfA, and swH1 primer/probe sets detect influenza A, swine influenza A, and influenza A/H1N1 2009, respectively. The RP primer/ probe set targets the human RNase P gene and serves as an internal positive control. The InfA, swInfA, and swH1 primer/probe sets must all be positive to confirm the presence of influenza A/H1N1 2009; specimens not positive for these three primer/probe sets were resolved with the FDA-approved xTAG® Respiratory Viral Panel v1 (Luminex-RVP). All testing was performed according to the manufacturer's instructions, and testing personnel were blinded to all test results. Using CDC-qPCR as the gold standard, Directigen-EZ test performance characteristics were determined. Two-tailed t-test was used for pair-wise comparison of continuous variables where

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Table 1 Age- and CT-stratified performance characteristics of Directigen EZ Flu A+B to detect pandemic influenza A/H1N1 2009. Age (y)

TP

TN

FP

FN

% Sn (95% CI)

% Sp (95% CI)

PPV

NPV

Combined ≥65 18–64 ≤17 11–17 6–10 3–5 ≤2 CT Combined 30.1–40 25.1–30 20.1–25 ≤20

103 2 30 71 15 30 15 11

496 131 226 139 49 36 18 36

0 0 0 0 0 0 0 0

120 4 60 56 16 25 13 2

46.2 33.3 33.3 55.9 48.4 54.6 53.6 84.6

(39.5–53) (6.3–72.9) (24.4–43.6) (46.8–64.7) (31.0–66.9) (40.6–68.0) (35.5–72.5) (56.6–97.2)

100 100 100 100 100 100 100 100

100 100 100 100 100 100 100 100

80.5 97.0 79.0 71.6 75.3 77.0 68.3 94.7

120 51 59 10 0

46.2 0.0 35.2 86.1 100

(39.5–53) (0–6.3) (25.4–45.2) (76.1–93.1) (70.1–100)

103 0 32 62 9

(99.3–100) (97.3–100) (98.5–100) (97.5–100) (93.5–100) (91.5–100) (82.7–100) (91.5–100)

TP = true positive; TN = true negative; FP = false positive; FN = false negative; Sn = sensitivity; Sp = specificity; PPV = positive predictive value; NPV = negative predictive value. The CT value is indirectly proportional to the quantity of nucleic acid in the specimen—the lower the CT value, the higher the concentration of target nucleic acid in the NPS specimen prior to amplification.

applicable. Statistical analysis was performed with Microsoft Office EXCEL 2010. Blyth-Still-Casella 95% confidence intervals were obtained for sensitivity and specificity with StatXact10 (Cytel Studio 10; Cytel Inc., Cambridge, MA, USA). Twenty-nine (n = 29) of 748 NPS specimens (3.8%) were positive for influenza A by Directigen-EZ and only the InfA and RP internal control primer/probe sets of the CDC-qPCR assay. These 29 specimens were confirmed as either seasonal influenza A H1 or seasonal influenza A H3 by Luminex-RVP and excluded from the data set to maintain alignment with the goal of this study. Of the remaining 719 specimens tested by CDC-qPCR, 496 were positive only for the RP internal control primer/probe set and deemed true negatives; 223 were positive for all 4 primer/probe sets and deemed true positives. Directigen-EZ yielded positive results for 103 of the 223 positive specimens. False-positive Directigen-EZ results were not observed. Overall Directigen-EZ sensitivity, specificity, positive predictive value, and negative predictive value were 46.2%, 100%, 100%, and 80.5%, respectively. Age-stratified sensitivity was 55.9%, 33.3%, and 33.3% for ≤17 y, 18–64 y, and ≥65 y categories, respectively. Sub-stratification of the ≤17 y group yielded Directigen-EZ sensitivity of 84.6%, 53.6%, 54.6%, and 48.4% for ≤2 y, 3–5 y, 6–10 y, and 11–17 y categories, respectively (Table 1). When stratified by crossing threshold (CT), Directigen-EZ sensitivity was 100%, 86.1%, 35.2%, and 0% for CT value categories of ≤20, 20.1–25, 25.1–30, and 30.1–40, respectively (Table 1). The relationship between patient age and virus quantity present in NPS specimens is depicted in Table 2. Mean CT values for the combined, ≥65 y, 18–64 y, and ≤17 y categories were not statistically different. Sub-stratification of the ≤17 y group showed an association between lower mean CT values and younger patient age,

Table 2 Association between age and quantity of influenza A/H1N1 2009 virus in NPS specimens based upon the CDC-qPCR assay. Age (y)

n

Category

Mean (SD)

Combined ≥65 18–64 ≤17 11–17 6–10 3–5 ≤2

21.9 72.2 38.5 7.8 13.7 7.9 4.2 1.2

(19.2) (5.9) (12.5) (4.2) (2.1) (1.4) (0.8) (0.7)

223 6 90 127 31 55 28 13

Mean CT value (SD) Influenza A/H1N1 2009

Internal control

26.7 27.9 27.2 26.4 27.1 26.5 26.3 24.8

29.0 (2.1) 30.1 (1.9) 29.4 (2.3) 28.8 (1.9) 29.4 (2.0) 28.6 (1.7) 28.8 (2.5) 27.6 (1.0)

(4.1) (2.1) (4.2) (4.1) (4.9) (3.9) (3.1) (4.1)

especially in the ≤2 y group. Although not statistically significant in our study, this association is congruent with published literature stating that younger children secrete higher quantities of virus than adults (CDC, 2011; Doller et al., 1992; Kaiser and Briones, 1999) Likewise, studies have shown that optimal RIDT results are obtained when specimens are collected within 72 hours of symptom onset, as this correlates to peak viral shedding (Doller et al., 1992; Kaiser and Briones, 1999). In our study, only 28% of patients were from clinical settings covered by the auspices of HIC approval. Therefore, duration of symptoms before NPS specimen collection could not be ascertained via chart review for the majority of patients in this study. Despite this limitation, we demonstrated that Directigen-EZ test performance was better in very young patients; this is congruent with published literature. Internal validation (data not shown) of the CDC-qPCR assay demonstrated that a delta CT (ΔCT) of 3.3–3.5 cycles corresponded to a 1.0 log10 change in target nucleic acid concentration over the entire linear range for each of the 4 primer/probe sets. The difference in CT values for the InfA, swInfA, and swH1InfA primer/probes targets at each dilution across the linear range differed less than 1.0 cycle. Influenza A/H1N1 2009 and internal control CT data from the CDCqPCR assay for true-positive, true-negative, and false-negative Directigen-EZ test results are shown in Table 3. For influenza A/H1N1 2009, the ΔCT for Directigen-EZ true positives and false negatives ranged from 5.5 to 6.1 cycles. This equates to an approximate 1.5–2.0 log10 higher concentration of virus in NPS specimens deemed Directigen-EZ true positive (mean CT = 23.8) versus false negative (mean CT = 29.3), establishing the cut-off range for the Directigen-EZ test to distinguish true positive from false-negative test results. These data were statistically significant for the ≤17 y, 18–64 y, and agecombined categories; the ≥65 y category did not reach statistical significance due to an insufficient number of specimens (n = 6). For the internal control, the ΔCT for Directigen-EZ true positives, false negatives, and true negatives for any age category ranged from 0.6 to 1.0 cycle; these differences were not statistically significance. This signifies that an equivalent amount of clinical material was obtained from each patient during the specimen collection process; likewise, specimen collection adequacy was well-controlled and had minimal impact on Directigen-EZ performance. To our knowledge, this is the first manuscript to determine the performance of the Directigen-EZ-RIDT under tightly controlled preanalytical conditions. We have verified that viral load and patient age are primary determinants of Directigen-EZ test performance. The impact that these determinants has, solely or synergistically in combination with duration of illness, on the performance of the

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Table 3 Directigen-EZ Flu A+B cut-off range using CDC-qPCR influenza A/H1N1 2009 and internal control mean CT values for true positive and false negative Directigen EZ results. Age (yr)

≤17

18–64

≥65

Combined

Internal controla

Influenza A/H1N1 2009

n CT mean ΔCT P value n CT mean ΔCT P value n CT mean ΔCT P value n CT mean ΔCT P value

(SD)

Directigen (TP)

Directigen (FN)

Directigen (TP)

Directigen (FN)

71 23.9 (2.6)

56 29.6 (3.3)

71 28.4 (1.7)

56 29.3 (2.2)

5.7 b0.0001 (SD)

30 23.1 (2.3)

0.9 N0.05 60 29.2 (3.3)

30 28.2 (1.8)

6.1 b0.0001 (SD)

2 28.1 (1.1)

1.0 N0.05 4 27.9 (2.6)

2 28.4 (0.3)

0.2 0.89 (SD)

103 23.8 (2.6)

4 27.8 (2.5) 0.6 N0.05

120 29.3 (3.2) 5.5 b0.0001

60 29.2 (2.1)

103 28.3 (1.7)

120 29.6 (2.2) 0.9 N0.05

n = number of specimens; ΔCT, Delta CT (Directigen false negative − Directigen true positive). a Mean CT (SD) Internal Control (RNase P) data for Directigen-EZ True Negative (n = 496) specimens: ≤17 y, 28.7(2.2); 18–64 y, 29.1(2.4); ≥65 y, 28.7(2.7); combined, 29.2 (2.6).

Directigen-EZ and other RIDTs under tightly controlled conditions still needs to be ascertained. References Al Johani SM, Al Balawi M, et al. Validity of two rapid point of care influenza tests and direct fluorecence assay in comparison of real time PCR for swine of origin Influenza virus. J Infect Public Health 2011;4(1):7–11. Centers for Disease Control and Prevention. CDC protocol of real time RTPCR for influenza A (H1N1). World Health Organization Website. http://www.who.int/csr/ resources/publications/swineflu/realtimepct/en/, 2009a. Centers for Disease Control and Prevention. Evaluation of Rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus - United States, 2009. MMWR Morb Mortal Wkly Rep 2009b;58:826–9. Centers for Disease Control and Prevention. Antiviral agents for the treatment and chemoprophylaxis of influenza – recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2011;60(1):1–24. Centers for Disease Control and Prevention. Evaluation of 11 commercially available rapid influenza diagnostic tests–United States, 2011–2012. MMWR Morb Mortal Wkly Rep 2012;61(43):873–6.

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