Validity of two rapid point of care influenza tests and direct fluorescence assay in comparison of real time PCR for swine of origin influenza virus

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1 Journal of Infection and Public Health (2011) 4, 7 11 Validity of two rapid point of care influenza tests and direct fluorescence assay in comparison of real time PCR for swine of origin influenza virus S.M. Al Johani a,b,, M. Al Balwi a,c, B. Al Alwan b, R. Al Hefdhi b, A. Hajeer a,d a College of Medicine, King Saud Bin AbdulAziz University for Health Science, Saudi Arabia b Division of Microbiology, Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh 11426, Saudi Arabia c Division of Molecular Biology, Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh 11426, Saudi Arabia d Division of Immunology, Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh 11426, Saudi Arabia Received 15 March 2010; received in revised form 25 September 2010; accepted 11 October 2010 KEYWORDS Rapid influenza diagnostics; DFA; RT-PCR; H1N1 Abstract Background: A novel swine origin influenza virus (S-OIV) is continue to spread worldwide and a global declaration of 2009 influenza pandemic was made by World Health Organization (WHO) June 2009, this along with approaching the winter season at the northern hemisphere, increase the interest to provide a quick, easy, affordable and available point of care testing for S-OIV. Objectives: To determine the performance of two rapid point-of-care (POC) tests for influenza virus as well as direct fluorescence assay for the detection of the recently emerged a novel swine origin influenza virus (S-OIV). Study design: A total of 143 respiratory samples which was submitted to Pathology and Laboratory Medicine at King AbdulAziz Medical City in Riyadh, Saudi Arabia from June 6th 2009 till June 28th All samples were tested in parallel using two rapid assays (BD Directigen EZ Flu ) and (TruFlu, Meridian ) as well as (Imagen Flu A/B DFA, Oxoid ) and compare it with RT-PCR. Each test s performed by different team, who were blinded for other team s result. Data gathered and we analyzed the analytical validity of each test. Corresponding author at: Division of Medical Microbiology, Department of Pathology and Laboratory Medicine, Mail Code 1122, P.O. Box 22490, King Abdulaziz Medical City, Riyadh 11426, Saudi Arabia. Tel.: x address: johanis@ngha.med.sa (S.M. Al Johani) /$ see front matter. Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved. doi: /j.jiph

2 8 S.M. Al Johani et al. Results: The analytical sensitivity of the two influenza antigen detection tests for S-OIV was very low in comparison with RT-PCR, BD Directigen EZ performance was better than TruFlu test with sensitivities of 20.6% and 9.7% respectively. DFA perform much better than POC tests with sensitivity of 32.35%, specificity of 99.08% and PPV, NPV of 90% and 81.20% respectively. Conclusion: The analytical sensitivity of the selected influenza A antigen detection tests for detection of S-OIV was very low, and should not be used to exclude S-OIV, DFA may be used as first line test especially during after hours or weekends, but negative results must confirmed by RT-PCR. Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved. 1. Introduction A novel swine origin influenza virus (S-OIV) was first identified in April The pandemic once declared there were major moves in most of the laboratory to find a rapid, easy, highly sensitive and specific diagnostic method. A novel real-time RT- PCR for S-OIV was set up in very short time and validated following industry-standard criteria [1,2]. Rapid diagnosis of influenza can facilitate timely clinical management decisions and applications of infection control precautions. Mainly because rapid influenza antigen tests provide a result in 30 min or less, and can serve as point of care tests at emergency room and triage areas. These tests are CLIA 88 regulated and are widely used for diagnosis of influenza in central, point-of-care, and physician office laboratories. Several rapid antigen tests are commercially available, some of which are able to distinguish between influenza A and B types. Rapid antigen tests are less sensitive in comparison to culture and RT-PCR. In seasonal influenza with sensitivity ranging between 50 and 80% in several studies [1,3 5]. In this study, we evaluated the validity of two rapid influenza A tests during the current S-OIV H1N1 outbreak and compare it to RT-PCR results at King Abdulaziz Medical City, In Riyadh, Saudi Arabia. 2. Materials and methods This is a cross sectional study of the validity of two different techniques for detection of H1N1 infection in 143 respiratory samples at KAMC during the Influenza H1N1 pandemic, from June 6, 2009 through June 28, All 143 respiratory samples (Nasopharyngeal swabs or Nasopharyngeal aspirates Table 1) collected from patient s with influenza like illness (ILI), all samples collected at employee health clinic for hospital employee and their dependant and all collected by registered nurse (RN), NO antiviral received by the patient prior to sample collection. Samples were tested in parallel using a real-time reversetranscriptase PCR (RT-PCR) kits along with TruFlu rapid influenza A and B, another rapid assay Directigen EZ (BD Medical; Sparks, Maryland ) test and Direct ImmunoFlourecence assay (DFA) (Imagen ). All specimens were collected from patients with influenza-like illness who met the World Health Organization and CDC s guidelines for screening and tested within 2 4 h of arrival to our laboratory, All samples were accessioned and stored at 2 C until tested [6] PCR method S-OIV is molecularly detected by reversetranscription polymerase chain reaction (RT-PCR) based assay. RT-PCR of S-OIV was performed at Molecular Biology Laboratory at our institute, who are blinded to other tests results (Table 2). Viral RNA was extracted on the QIAsymphony SP system from 400 L nasopharyngeal aspirates Using the QIAsymphony Virus/Bacteria Kits (Qiagen, Hamburg, Germany ). The extracted viral RNA was eluted into 60 L with elution buffer. A 10 L of RNA was reverse-transcribed to cdna using Transcriptor First Stand cdna Synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany ), following the manufacturer recommendation. The resultant cdna is then amplified and screened LightMix Kit InfA M2 for detection of presence of influenza A matrix protein, all positive samples fro matrix protein then will be tested with specific primers and probes for H1N1 using LightMix InfA swine H1 kit (TIB MOL- BIOL GmbH, Berlin, Germany) and following the manufacture recommendation. PCR was performed in a total volume of 20 L in glass capillaries. The reaction mixture used in each PCR consisted of 5 L of cdna (specimen/positive control/negative control), 2 L of the primers/probe mixture, 2 L of the Roche FasterStar DNA Master Hybridization Probes Kit reagent, 2.4 L of MgCl 2 (25 mmol/l), and 8.6 L od PCR-grade water.

3 Performance characteristics of two rapid influenza diagnostic tests 9 Table 1 Number and percentage of samples tested positive or negative in the influenza tests. Samples included 13 nasopharyngeal aspirates, 127 nasopharyngeal swabs and 3 throat swabs. Result Test used RESP DFA /Imagen TruFlu RT-PCR EZ FLU POSITIVE Samples Number 12 (8.4) 5 (3.5) 34 (23.8) 8 (5.6) NEGATIVE Samples Number 131 (91.6) 138 (96.5) 109 (76.2) 135 (94.4) The amplification conditions consisted of one denaturation/activation cycle of 10 min at 95 C and 50 cycles of three-temperature amplification. Each cycle consisted of 95 C for 5 s, 62 C for 5 s, and 72 C for 15 s with a single fluorescent acquisition step at the 62 C hold. This was followed by a melting curve analysis of 95 C for 10 s, 40 C for 20 s, and a slow ramp (0.2 C/s) to 85 C with continuous fluorescent acquisition. The presence of H1N1 genotype is also confirmed by the melting curve analysis using the Roche lightcycler 2.0 instruments Rapid antigen assays Rapid antigen assays were used according to the manufacturer s instructions on 100 L of freshly collected original sample from patients with ILI. Two positive controls of known seasonal influenza and S-OIV included in each run as well as one known negative control. The rapid antigen assays was performed by Microbiology Technologists who are blinded for other tests result Immuno-fluorescence assay A slide was coated by 25 l of influenza A virus reagent, then Incubated for 15 min at 37 Cina moist chamber. After the incubation period, slides washed with Puffer Solution and placed into the PBS bath. Then we drain off PBS and allow the slides to air dry at room temperature (15 30 C). Finally, we added one drop of IMAGEN TM mounting fluid to the centre of each well and place Table 2 Test validity compare to RT-PCR. Senstivity Specificity PPV NPV TruFlu Directigen EZ Imagen Respiratory DFA a cover slip over the mounting fluid and specimen ensuring that no air bubbles are trapped. Then we examine the entire 6 mm well area containing the stained specimen using an fluorescence microscope. Fluorescence should be visible at magnifications. The immuno-fluorescence assay was performed by Virology Technologists who are blinded for other tests result. 3. Results A total of 143 respiratory samples were received in the laboratory in Viral Transport Medium (VTM), all were tested in parallel by using two different rapid kits, DFA and RT-PCR, each team of technologists were blinded to other team s results. All the data were entered in an Excel TM sheets and analyzed. Overall, all tests were able to detect S-OIV at variable sensitivity. For POC testing, BD Directigen test demonstrated marginally greater sensitivity than the TruFlu test. The sensitivity, specificity, PPV and NPV for TruFlu test; in comparing to RT-PCR; are 9.7%, 98.2%, 60% and 77.5% respectively. While the sensitivity, specificity, PPV and NPV for BD Directigen is: 20.6%, 99.08%, 87.5% and 80% respectively. DFA showed superior results than the POC tests with sensitivity of 32.35%, specificity of 99.08%, as well as 90% and 81.20% for positive and negative predictive values respectively comparing to RT-PCR. Viral infectivity and RNA load data for viruses at the detection limit of the rapid test kits, suggested that both the BD Directigen and TruFlu tests were less sensitive for the detection of A novel swine origin influenza virus (S-OIV) than for human seasonal strains. 4. Discussion Rapid Point of Care (POC) Influenza tests currently on the market are designed to detect influenza type A, type B or both. They can distinguish influenza A from influenza B, but cannot distinguish S-OIV from seasonal strains of Influenza [7].

4 10 S.M. Al Johani et al. As the S-OIV is a type A influenza virus; so if the result is positive for influenza A, the patient may be infected with S-OIV or with seasonal strains of influenza A [7]. However, current data from the US Centers for Disease Control and Prevention (CDC) indicate that currently, 99% of the circulating influenza viruses in the United States are S-OIV H1N1 [8]. Thus, although it is not possible to confirm S-OIV infection with an influenza rapid POC test, a positive rapid POC test result for influenza A is assumed to be due to infection with S-OIV H1N1 [7]. Routine testing for S-OIV using rapid POC tests is not recommended by the CDC because the sensitivities of the currently available rapid POC tests for the detection of S-OIV are quite poor. Various studies have shown detection rates between 11% and 70% [9,10,12,13]. This means that the rapid POC test may fail to detect S-OIV in 30 90% of cases. It is critical for all health care workers (HCW) to be aware that a negative result on an influenza rapid POC test does not rule out S-OIV infection. For this reason, the CDC recommends that management of patients with suspected S-OIV infection should be based on symptoms and underlying risk factors rather than the result of a rapid flu test [8]. Physicians should also be wary of the indiscriminate use of influenza rapid tests during a period when influenza is not circulating at high levels. This is because the specificity of these assays is not as high as for culture or RT-PCR. The positive predictive value of a test for an infectious disease such as influenza depends on the specificity of the test and on the prevalence of the disease in the population tested [7]. If influenza prevalence is high, the positive predictive value of a rapid test is increased, and thus a positive test is more likely to represent a true positive. However, during periods when influenza activity is low, such as during most summer months, a positive result on a rapid test is much more likely to represent a false positive. During periods of low prevalence, physicians who require a definitive diagnosis should order tests with high levels of specificity, such as culture or RT- PCR, because false-positive results are significantly less likely [7,9 11,14 16]. 5. Conclusion Rapid antigen tests, DFA and the RT-PCR test all detected the S-OIV strain, but with variable sensitivity. The RT-PCR test provided the best diagnostic option as it demonstrated superior sensitivity for the detection of influenza A matrix protein and differentiate between S-OIV and other influenza A subtyping, but it is very expensive and more labor intensive over other tests. Early diagnosis of infection can assist in the rapid treatment. However the tests are significantly less sensitive than PCR assays and as such, negative results should be verified by other laboratory tests. Conflict of interest statement Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Panning M, Eickmann M, Landt O, Monazahian M, Olschläger S, Baumgarte S, et al. Detection of influenza A(H1N1)v virus by real-time RT-PCR. Euro Surveill 2009;14(September (36)), pii: [2] Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009;360: [3] Loeffelholz MJ. American Society for Microbiology; Sentinel laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases; Avian Influenza A H5N1; December 2008 [accessed ]. [4] Al Johani S. Swine influenza H1N1; is your laboratory prepared? Saudi Med J 2009;30(7). [5] Cheng CK, Cowling BJ, Chan KH, Fang VJ, Seto WH, Yung R, et al. 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