Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 1, 2011, pp. 19-21

Indian Journal of Medical Microbiology, Vol. 29, No. 1, January-March, 2011, pp. 19-21

Original Article

BinaxNOW^® - An immunochromatographic test for the diagnosis of human influenza viruses: Comparison with viral culture and polymerase chain reaction

S Bhattacharya, H Osman

HPA West Midlands Public Health Laboratory, Birmingham Heartlands Hospital, United Kingdom

Correspondence Address: H Osman, HPA West Midlands Public Health Laboratory, Birmingham Heartlands Hospital , United Kingdom, husam.osman@heartofengland.nhs.uk

Date of Submission: 20-Oct-2009
Date of Acceptance: 08-Sep-2010

Code Number: mb11005

PMID: 21304189

DOI: 10.4103/0255-0857.76518

Abstract

Keywords: Influenza, rapid antigen test, sensitivity

Introduction

Rapid detection of influenza viruses in clinical specimens has clinical and infection control implications. ^[1] Its identification by direct immunofluorescence (DIF) is labour intensive and insensitive. This study evaluated the performance of BinaxNOW^® kit (Binax, Inc., Scarborough, ME, USA) and compared its analytical performance with viral culture and reverse transcriptase-polymerase chain reaction (RT-PCR).

Materials and Methods

Study design

In season-1 (winter of 2004-2005), a retrospective study of 35 respiratory samples (bronchoalveolar lavage 2, nasopharyngeal aspirate (NPA) 8, nose + throat swab 7, nose swab 1, throat swab 10, tissue 2, and unspecified respiratory secretion/swab 5), positive for a variety of human influenza viruses by culture, were tested by Binax kit. Samples were stored at −20ºC till the time of testing. In season-2 (winter of 2005-2006), a prospective study of 45 samples (nasopharyngeal aspirate 1, nose + throat swab 30, nose swab 4, throat swab 8, and unspecified respiratory secretion/swab 2) collected from patients with influenza-like illness were tested by viral culture, Binax and RT-PCR. All samples were collected from patients after the onset of respiratory symptoms in the acute phase of illness. The study was part of the regular kit/test evaluation work carried out by the departmental scientific/technical team, and the data were reviewed by the research and development section.

Techniques

Viral culture was performed using rhesus monkey kidney and primary liver carcinoma cell line as per the established local protocol. After incubation at 37ºC, influenza viruses were detected by haemadsorption test using 0.4% guinea pig erythrocytes. ^[2] DIF (Imagen respiratory screen kit; Dako Corporation, Carpinteria, CA, USA) was performed on cells giving positive haemadsorption test to confirm virus isolation. BinaxNOW Influenza A and B kit was used following the manufacturer′s instructions. The typing of the isolates was performed at HPA laboratory at Colindale, UK.

Roche MagNAPure (Roche Diagnostics, Manheim, Germany) was used for automated total nucleic acid extraction. The RT-PCR were performed on ABI 7500 fast real-time PCR (Applied Biosystems, Cheshire, UK) using a multiplex format. ^[3] The one-step RT-PCR thermal profile was as follows: 50ºC for 15 minutes, 95ºC for 2 minutes, 95ºC for 15 seconds repeated 45 cycles, and then 60ºC for 40 seconds.

Quality control (QC) procedures were rigorously followed routinely as per regular departmental QC activities. This included incubating cell lines with positive, negative and cell controls. PCRs were performed with each sample spiked with Borne Mosaic virus as internal amplification control. All the tests were performed in a CPA (Clinical Pathology Accreditation, UK) accredited laboratory which regularly participates in National External Quality Assessment Service (NEQAS) and Quality Control Molecular Diagnostics (QCMD) exercises.

Statistical analysis

The software from http://www.graphpad.com was used to perform statistical tests. Unpaired t test was used to compare the differences in the mean age at baseline patient characteristics; in all other cases, including comparison of the differences in the sample types between the two seasons, source of samples (outbreak or sporadic cases), comparison of gender differences in sample numbers, positivity rates for influenza virus A or B in the two seasons, the Fisher′s exact test was used.

Results

Binax kit failed to detect 13 and 20 culture positive samples in the two seasons. In season-2, the number of samples positive by PCR, culture, and culture as well as PCR was 29, 26 and 23, respectively. Also, 6 samples were PCR positive but culture negative, and 3 samples were culture positive but PCR negative. Altogether, in season-2, Binax failed to detect 25 samples, which were either culture or PCR positive [Table - 1]. The specificity of Binax was high (100 and 94.7% with influenza types A and B, respectively). Binax negative samples had significantly higher CT values (low viral load) compared to Binax positive samples (CT values 30.2 and 23.9, respectively; P = 0.0029) [Figure - 1].

Discussion

Discrepancy between pre- and post licensure performance as seen here has been observed in previous studies as, for example, in Australia, New Zealand and USA, where influenza B sensitivity was found to be 30, 23 and 35.9%, respectively, ^[4],[5],[6] with the exception of one USA study (sensitivity 100%). ^[7] Binax influenza A sensitivity was better with reports of 73, 78.3, 73%, respectively, ^[4],[6] with the exception of a New Zealand study (sensitivity 59%). ^[5] Specif[icity of Binax was high with all studies like the present one (95-100%). ^{[4],[5],[6],[7]} Sensitivity problems with other diagnostic kits such as BD Directigen^™ (Becton Dickinson, NJ, USA) ^{[4],[5],[7],[8]} and Remel Xpect (Remel, Santa Fe, NM, USA) ^[6] have been reported. Binax was found to have a poor negative predictive value, making it difficult to exclude the diagnosis in cases of a negative result. A limitation of this study is the relatively small sample size. Sample numbers were based in this study on the number of samples positive for influenza viruses in season-1. A comparable number of samples were included in the study in season 2.

The drop in sensitivity of Binax in season-2 could be due to the change in predominant circulating strain ^[6] [season-1: Influenza-A/California/7/2004 (H3); season-2: Influenza-B/Tehran/80/2002], low viral load in the specimens, ^[9] difference in patient profile ^[6],[10] (more samples from infants and >65 years in season-1) or sample profile ^[6] (more nasopharyngeal aspirates in the season-1 and more nose and throat swab in season-2). Patients in the extremes of age, possibly because of relatively low immunity, have higher viral load in respiratory samples and virus in excreted for a longer duration. The timing of specimen collection could also affect the sensitivity, since the viral load in respiratory secretions is often highest during the early stages of the disease. For most of the paediatric (≤12 years) patients (6/35 in season-1 and 16/45 in season-2), the samples are usually collected at admission. However, the same is not necessarily true for adult patients. It is also possible that vaccinated individuals would have a lower viral load and make the rapid assay less sensitive. We do not know how many patients in our study were vaccinated.

Significant difference in clinical sensitivity of BinaxNOW and RT-PCR for influenza viruses has been recently reported (53% versus 100%, respectively). ^[11] It was also reported in the same study that eight true positive samples did not become reactive by Binax until 30 minutes, and thus were counted as negative. In the present study test, readings for Binax were taken after 15 minutes as per manufacturer′s instructions, so it is not possible to comment if some of the falsely negative samples could have become positive after extended incubation at room temperature. Again, low viral load in a sample could be due to sampling factors (inadequately or inappropriately taken samples) rather than patient factors (e.g. very early or late presentation).

Finally, results from the Centers for Disease Control and Prevention (CDC) have shown that although the rapid influenza diagnostic tests were capable of detecting the novel H1N1 virus from respiratory specimens containing high viral loads (as indicated by low RT-PCR cycle threshold values), the overall sensitivity was low (40-69%) among all specimens tested and declined substantially as viral load decreased. Therefore, patients with illnesses compatible with novel influenza A (H1N1) virus infection but with negative antigen test results should be considered for empirical treatment based on the level of clinical suspicion and illness severity. ^[12]

To conclude, performance evaluation of diagnostic kits over more than one season of influenza could be complicated by differences in patient or sample profile. Information in kit inserts regarding pre-licensure sensitivity or predictive values may not be indicative of post licensure performances in clinical settings. We found the Binax tests for influenza having good specificity and positive predictive values but poor in negative predictive values and sensitivity. Relying on these kits to exclude the diagnosis would be dangerous and may lead to serious clinical and infection control problems. The advantages of Binax include ease of use, less training requirements, short turnaround time. These properties could be useful for a low-tech out-of-hours service in virology taking into account the limitations of sensitivity.

References

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