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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 1  |  Page : 63-67

A comparison of three diagnostic platforms for the detection of influenza A and B in children


1 Department of Paediatrics and Child Health, Aga Khan University, Karachi, Pakistan
2 Aga Khan Medical College, Aga Khan University, Karachi, Pakistan
3 Department of Paediatrics and Child Health; Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan

Date of Web Publication5-Mar-2018

Correspondence Address:
Dr. Najeeha Talat Iqbal
Department of Paediatrics and Child Health and Biological and Biomedical Sciences, Aga Khan University, Stadium Road, P.O Box 3500, Karachi
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_107_17

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  Abstract 


Background: Viral flu is the predominant cause of hospitalization in young children, which invariably leads to enhanced morbidity and mortality in children in developing countries. Initial treatment of viral flu is based on presumptive diagnosis. Bedside testing is not common in clinical settings because of variable sensitivity and specificity of rapid tests in different settings. Methods: To address this issue, we evaluated the performance of Binax influenza A/B rapid testing kit against two robust molecular platforms (quantitative real-time polymerase chain reaction [qRT-PCR] and TaqMan array card [TAC]) in 24 nasopharyngeal (NP) swabs, collected from children under 5 years of age. Results: Binax was found to be less sensitive (56%), but 100% specific compared to qRT-PCR (100%) and TAC (>100%). Using TAC cards, 75% of samples were found to be coinfected with other bacterial and viral targets. Conclusion: Binax flu is suitable for bedside testing in clinical and community settings. The negative results of Binax should be interpreted with caution and confirmed by rapid molecular tests.

Keywords: Human influenza, immunochromatography, real-time polymerase chain reaction


How to cite this article:
Kabir F, Tariq M, Aziz F, Rizvi SI, Qureshi S, Ali A, Iqbal NT. A comparison of three diagnostic platforms for the detection of influenza A and B in children. Biomed Biotechnol Res J 2018;2:63-7

How to cite this URL:
Kabir F, Tariq M, Aziz F, Rizvi SI, Qureshi S, Ali A, Iqbal NT. A comparison of three diagnostic platforms for the detection of influenza A and B in children. Biomed Biotechnol Res J [serial online] 2018 [cited 2020 Feb 24];2:63-7. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/63/226574




  Introduction Top


Viral influenza is the leading cause of deaths among children in developing countries. It is most common cause of admission of infants in emergency rooms for the treatment of seasonal flu.[1] Continuous evolution of new viral strains renders previous vaccines ineffective. Early diagnosis facilitates the initiation of antiviral treatment.[2],[3] This, in turn, reduces risk of transmission,[4] unnecessary usage of antibiotic, and length of hospital stay.[5] Molecular diagnostic tests are accurate, albeit not suitable as point-of-care test (POC). Rapid test is suitable for bedside testing; it requires a high viral load for positivity.[6]

This study evaluated the performance of ICT-BinaxNOW kit (Alere Binax, Portland, ME), against quantitative real-time polymerase chain reaction (qRT-PCR) (CDC Ref. # I-007-05, Version 2009) and TaqMan array card (TAC) assay (Applied Biosystems, Foster City, CA) for influenza A and B viral testing in children less than 5 years of age. BinaxNOW kit is anin vitro immunochromatographic assay technique, used for the qualitative detection of influenza A and B nucleoprotein antigens in nasopharyngeal (NP) swab/wash/aspirate specimens. Within available molecular platforms, TAC is proving to be a valuable tool for multiple pathogen detection. It is a 384-well microfluidic array comprising of precoated/customized dried-down primers and probes as a singleplex qPCR reaction, used for simultaneous detection of up to 100 targets from a single specimen. It has been employed and evaluated previously for the identification of respiratory pathogens, enteropathogens,[7] and biothreat agents.[8] The clinical application of TAC is limited for bedside testing as it demands high technical expertise and infrastructure. In this study, we confirmed a low but invariable sensitivity of Binax from both clinical and community samples compared to high throughput and robust molecular tests of q-RT PCR and TAC assay.


  Methods Top


Study samples (n = 24) were obtained from two study cohorts: Aetiology of Neonatal Infection in South Asia (ANISA) and Global Research Initiative Program (GRIP) study, in semi-urban and rural area of Karachi, between 2009 and 2014, respectively. Children with upper respiratory symptoms were enrolled in the original studies. Study groups comprised of neonates (n = 8), infants (n = 10), and children (n = 6) under five. Nasopharyngeal/oropharyngeal (NP/OP) swabs were collected and transported to research laboratory using viral transport media under cold chain maintained at 2°C–8°C and stored in DMSO at −80°C until further use.

Binax testing was performed as per the manufacturer's protocol by visually assessing the appearance of colored bands within 15 min [Supplementary Figure 1]. Total nucleic acid (TNA) was extracted from automated nucleic acid extractor (Roche MagNA pure compact, Roche Diagnostics, Germany), before processing for qRT-PCR and TAC assay. Briefly, RT-PCR was performed as per CDC protocol using ABI 7500 real-time PCR (RTPCR) machine (Life Technologies, USA). 25 μL reaction cocktail was prepared by adding 20 μL QuantiTect Probe PCR (Qiagen) mix containing influenza A/B specific primers and probes were added to the 5 μL template TNA. The cycling condition for qRT-PCR was 50°C for 30 min, Taq activation for 2 min at 95°C, and 45 cycles of 95°C for 15 s and an annealing-extension step at 55°C for 30 s. The TAC was performed as described by Diaz et al.[8] In a 100 mL reaction, 50 mL Quanta master mix (VWR, USA) and 50 mL of TNA were used, the card was spin at 1200 rpm for 1 min each (Thermo Fisher Scientific, Germany), card was sealed with the help of array card stacker sealer (Applied Biosystems, USA), and the upper portion of the card was trimmed and ran on ViiA7 RTPCR system (Applied Biosystems, USA). The cycling condition was 45°C for 10 min, 94°C for 10 min, followed by 45 cycles of 94°C for 30 s, and 60°C for 1 min. The cutoff for positive test was any cycle threshold (Ct) value before 40 cycles and appearance of S-shaped sigmoidal curve [Figure 1]. The TAC format was customized as per ANISA protocol.[8] [Supplementary Figure 2] illustrates the layout of TAC.
Figure 1: Screenshot of TaqMan array card run file, using QuantStudio real--time polymerase chain reaction software version 1.2

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  Results Top


Of 24 NP/OP tested, Binax had 37.5% (9/24) positive and 62.5% (15/24) negative test. Of 37.5% positive test, 29% were positive for influenza A and 8.3% positive for influenza B. Compared to Binax, qRT-PCR was positive for 66% (16/24) of test samples; of these, 42% and 25% were positive for influenza A and influenza B, respectively [Supplementary Table 1]. On the whole, qRT-PCR detected additional 7 samples that were missed by Binax test. The mean Ct value was 29.79 ± 5.3 (range = 23.45–40.9). TAC assay found to be more sensitive compared to qRT-PCR and detected three additional samples that were missed by qRT-PCR. In TAC assay, 50% (12/24) samples were positive for influenza A and 29% (7/24) were positive for influenza B. The combined sensitivity of Binax for both influenza A and B was 56% and specificity was 100%, keeping qRT-PCR as a gold standard. The sensitivity for influenza A was 70% (95% confidence interval: 34.75–93.33) and influenza B was 33% (95% CI: 4.33–77.72). The combine sensitivity of TAC was >100% compared to qRT-PCR for both influenza A and influenza B. [Table 1]a shows the comparison of Binax influenza A with qRT-PCR. The sensitivity of Binax was 70.0% and specificity was 100%, with corresponding positive and negative predictive value (PPV and NPV) of 100% and 72.7%, respectively. The sensitivity of influenza B was much lower without affecting the specificity of the test (sensitivity [33.3%], specificity [100%], PPV [100%], and NPV [66.6%]). [Table 1]b shows the comparison of qRT-PCR with TAC assay. The sensitivity of qRT-PCR for influenza A was 83.3% and influenza B was 85.7% with a corresponding specificity of 100% for both targets. The association of Binax and qRT-PCR was significant for influenza A (P = 0.004; Fisher's exact test) but not for influenza B (P = 0.165; Fisher's exact test), [Table 1]a. Compared to Binax, TAC was significantly associated with qRT-PCR platform for both influenza A (P = 0.003) and influenza B (P = 0.0152) [Table 1]b. This correlates well for the positivity obtained from both platforms.

Table 1a: Comparison of Binax versus quantitative real-time polymerase chain reaction (n=24)a

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Table 1b: Comparison of quantitative real-time polymerase chain reaction versus TaqMan array card (n=24)a

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A quantitative comparison was performed between Ct values obtained from qRT-PCR and TAC assay. The mean Ct value of qRT-PCR was 30.25 compared to 23.91 of TAC (paired t-test; P = 0.017). This shows higher sensitivity of the TAC assay for sample with low viral load [Figure 2].
Figure 2: Line graph shows the comparison of cycle threshold values in quantitative real-time polymerase chain reaction and TaqMan array card. The sold horizontal bars indicate mean cycle threshold values from the two platforms. Paired t-test was applied for comparison of mean in the 16 positive samples by both platforms

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The unique feature of TAC assay is a multiple pathogen detection along with influenza A and B target [Figure 3]. Among bacterial targets, Streptococcus pneumoniae was the most common (37%) in all age groups followed by Klebsiella pneumoniae (20.8%). Among viral targets, respiratory syncytial virus, enterovirus, rhinovirus, and cytomegalovirus (CMV) were mostly present in younger children while rubella virus, rhinovirus, and CMV were the most common viral targets in neonates and infants along with influenza A/B [Table 2]. In five influenza A/B-negative samples, TAC assay was able to detect rubella, rhinovirus, and S. pneumoniae as additional pathogenic targets in neonates [Table 2].
Figure 3: Bar graph shows the proportion of additional bacterial and viral targets detected by TaqMan array card along with FluA/FluB. The X-axis shows the % positivity for each target. (STPN: Streptococcus pneumoniae, KLPN: Klebsiella pneumoniae, CYMV: Cytomegalovirus, RHIV: Rhinovirus, ADEN: Adenovirus BOP: Bordetella pertussis, ECHS:  Escherichia More Details coli/Shigella species, ENTV: Enterovirus, GBST: Group B Streptococcus, RUBV: Rubella virus, RESV: Respiratory syncytial virus, URUP: Ureaplasma parvum/urealyticum)

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Table 2: Distribution of coinfection in different age groups

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  Discussion Top


We compared the performance of three diagnostic platforms for the detection of influenza A and B in children with respiratory symptoms. In addition, we detected viral and bacterial coinfection other than influenza A/B using a highly sensitive TAC assay.

Although Binax allows prompt initiation of antiviral therapy that limits the injudicious use of antibiotics in viral influenza, the lower detection limit of the test is also problematic in case of lower viral load. Such rapid tests are useful in surveillance and POC testing in pandemic situations. The downside of rapid diagnostics test is its lower sensitivity and greater false negativity rate (~44%) as documented by other studies.[9]

Results showed a higher specificity (100%) but lower sensitivity (56%) of Binax when compared to molecular methods. Moderate false-negative rate of Binax may lead to incorrect diagnosis and cause delay in appropriate therapy.[10] These findings are comparable to other studies that have demonstrated combined sensitivity of Binax ranging from 11.1% to 60.3% and specificity ranging from 93.6% to 100.0%, using qRT-PCR (as a gold standard method).[11]

As recommended by the CDC, results from rapid tests should always be interpreted in the broader context of the circulating influenza strains, pandemicity, clinical suspicion, severity of illness, and risk for complications due to superimposed bacterial infection. Negative results should be further evaluated with more sensitive testing.[12]

An interesting finding in our study was the predominance of S. pneumoniae and K. pneumoniae coinfection in children with and without influenza. The dual infection is the main cause of mortality as reported in the 2009 H1N1 pandemic.[13] In this context, TAC platform has application in the diagnosis of flu infections in pandemic areas with high risk of dual bacterial and viral infections. While TAC has been used quite extensively for the detection of diarrheal pathogens in multicenter studies,[7] it also showed a comparable sensitivity of TAC with qRT-PCR for enteropathogen detection. In a validation study, TAC assay showed a sensitivity ranging between 92% and 100% for influenza A/B. The sensitivity for other respiratory pathogen was lower compared to individual RTPCR.[14] Despite its limited use as POC test, TAC assay may be used as complimentary test to confirm the presence of coinfection in complicated cases of pneumonia. High-throughput feature and higher sensitivity of TAC assay are suitable for diagnostic laboratories apt with trained staff and equipment facility.


  Conclusion Top


In our study, Binax demonstrated low-to-moderate sensitivity for influenza A and B, which is comparable to previously reported results in meta-analysis with pooled sensitivity of 68%–81%.[15] It still remains as a choice of test for rapid diagnosis and POC testing, but negative results should be interpreted with caution and further testing is recommended as per CDC guidelines.

Acknowledgments

We gratefully acknowledge the study participants and their families, contributions of students of UGME Research Module Class 2020, Ms. Raima Hashmi and Ms. Mahrukh Amir toward conducting experiments and laboratory activities, and Dr H. R. Ahmed for proofreading. We would also like to acknowledge the pediatric infectious disease research laboratory staff, project field staff, and administration for their dedicated services and assistance provided in conducting the study.

Financial support and sponsorship

Funding support from Aga Khan Medical College Research module, ANISA and GRIP study is also acknowledged for sample collection and processing. We gratefully acknowledge Drs. Zulfiqar Bhutta and Anita Zaidi for providing samples from ANISA study.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Liu J, Kabir F, Manneh J, Lertsethtakarn P, Begum S, Gratz J, et al. Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: A multicentre study. Lancet Infect Dis 2014;14:716-24.  Back to cited text no. 7
    
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Diaz MH, Waller JL, Napoliello RA, Islam MS, Wolff BJ, Burken DJ, et al. Optimization of multiple pathogen detection using the TaqMan array card: Application for a population-based study of neonatal infection. PLoS One 2013;8:e66183.  Back to cited text no. 8
    
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Fuenzalida L, Blanco S, Prat C, Vivancos M, Dominguez MJ, Mòdol JM, et al. Utility of the rapid antigen detection BinaxNOW influenza A and B test for detection of novel influenza A (H1N1) virus. Clin Microbiol Infect 2010;16:1574-6.  Back to cited text no. 11
    
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Centers for Disease Control and Prevention. Guidance for Clinicians on the Use of Rapid Influenza Diagnostic Tests for the 2010-2011 Influenza Season; 2010.  Back to cited text no. 12
    
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Palacios G, Hornig M, Cisterna D, Savji N, Bussetti AV, Kapoor V, et al. Streptococcus pneumoniae coinfection is correlated with the severity of H1N1 pandemic influenza. PLoS One 2009;4:e8540.  Back to cited text no. 13
    
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Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ, et al. Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol 2011;49:2175-82.  Back to cited text no. 14
    
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Babin SM, Hsieh YH, Rothman RE, Gaydos CA. A meta-analysis of point-of-care laboratory tests in the diagnosis of novel 2009 swine-lineage pandemic influenza A (H1N1). Diagn Microbiol Infect Dis 2011;69:410-8.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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