Comparison of Becton Dickinson Directigen EZ Flu A+B Test against the CDC Real-Time PCR Assay for Detection of 2009 Pandemic Influenza A/H1N1 Virus

Pandemic influenza A/H1N1 2009 virus was first reported in Mexico during April 2009, followed by a rapid spread to many countries (10). As of 4 October 2009, >375,000 laboratory-confirmed cases and >4,500 deaths had been reported to the World Health Organization (10). The 2009 influenza season in the southern hemisphere was characterized by a predominance of this strain, which to date is the same pattern observed in the United States during the early 2009-2010 influenza season (3).

Although performance characteristics of several commercial rapid immunodiagnostic tests (RIDT) for the detection of seasonal influenza viruses are known (6), only limited data have been published regarding the ability of RIDT to detect pandemic influenza A/H1N1 2009 virus (1, 4, 5, 7-9). Of these comparisons, two included an evaluation of the Directigen EZ Flu A+B immunoassay (Becton Dickinson, Sparks, MD). In these two reports, the reported sensitivities were 46.7 and 49% (1, 9). To date, there are no reported data comparing the Directigen EZ Flu A+B immunoassay with the real-time PCR (RT-PCR) assay developed by the Centers for Disease Control and Prevention (CDC) to detect pandemic influenza A/H1N1 2009 virus. This communication is a report of such a comparison using fresh, clinical nasopharyngeal wash (NPW) specimens.

A total of 231 NPW specimens collected using the N-Pak nasopharyngeal aspiration kit (M-Pro, Annandale, MN) were submitted to the Clinical Microbiology Laboratory at Denver Health Medical Center for rapid influenza testing during August, September, and October. Of these, six were found by RT-PCR to be seasonal influenza viruses and were eliminated from the comparison. No swabs were submitted for testing. Directigen EZ Flu A+B RIDTs were performed 7 days per week between the hours of 0700 and 2300. Specimens received after 2300 h were refrigerated, and testing was performed the next morning. All tests were performed according to the manufacturer's instructions in the package insert (2). Aliquots of all specimens were refrigerated, saved, and sent daily Monday to Friday via courier to the Laboratory Services Division of the Colorado Department of Public Health and Environment. Testing for pandemic influenza H1N1 2009 virus was performed using the CDC-developed RT-PCR assay. This protocol currently operates under Emergency Use Authorization (EUA) status granted by the FDA and is designed to detect all influenza A viruses, swine strains of influenza A virus, and H1N1 2009. All three targets must yield positive findings for a patient specimen to be considered positive.

The results are shown in Table 1. Of 225 NPW specimens, 80 (35.6%) were positive and 145 (64.4%) were negative by RT-PCR. Of the 80 specimens that tested positive by RT-PCR, 39 tested positive by RIDT, for a test sensitivity of 48.7% and a positive predictive value of 88.6%. Of the 145 specimens that tested negative by RT-PCR, 140 tested negative by RIDT, for a test specificity of 96.5% and a negative predictive value of 77.3%. The overall agreement between the two assays was 179/225 (79.5%).

TABLE 1.

BD Directigen EZ Flu A+B compared to CDC RT-PCR for detecting pandemic influenza A/H1N1 2009 virus

RT-PCR resulta (nb)	No. of specimens with BD Directigen result		Sensitivityc	Specificityd	PPVe	NPVf
	Positive	Negative
Confirmed positive (80)	39	41	39/80 (48.7)		39/[39 + 5] (88.6)
Confirmed negative (145)	5	140		140/145 (96.5)		140/[140 + 41] (77.3)

^aAs determined by CDC RT-PCR.

^bn, no. of specimens with result.

^cNo. positive by BD Directigen/no. RT-PCR positive (%).

^dNo. negative by BD Directigen/no. RT-PCR negative (%).

^ePPV, positive predictive value. Percentage is given in parentheses.

^fNPV, negative predictive value. Percentage is given in parentheses.

Considerable attention has been given to clinical laboratory testing for the pandemic influenza A/H1N1 2009 virus. Much of this attention has focused on RIDTs, which have been demonstrated previously to have lower sensitivity than RT-PCR, viral culture, and other methods in the detection of seasonal influenza (6). In published reports regarding the use of RIDT for detecting pandemic influenza A/H1N1 2009 virus, the performance characteristics of several commercial RIDTs have been reported to be low. Of note, however, the multiplicity of methods evaluated, different types of specimens tested, lack of comparison against a consistent gold standard assay, and (in some reports) absence of information regarding the types of RIDTs used make it difficult for potential users to compare these assays.

To our knowledge, no study to date has compared the performance characteristics of RIDT with the CDC RT-PCR for the detection of pandemic influenza A/H1N1 2009 virus using exclusively fresh clinical NPW specimens. Our findings are consistent with those reported in the literature, confirming that this immunoassay is specific but insensitive in the detection of this strain of influenza virus. What diagnostic role RIDTs may play during this and subsequent influenza seasons depends on how they are used. For example, when a clearly predominant strain of influenza virus is circulating in a community, these tests may be useful as initial screening tests, although negative test results in cases with a high clinical suspicion of influenza should be followed up by use of a more sensitive test, such as a CDC or other RT-PCR.