Comparative Evaluation of Effectiveness of IAVchip DNA Microarray in Influenza A Diagnosis

The paper describes comparative evaluation of IAVchip DNA microarray, reverse transcription PCR (RT-PCR), and real-time RT-PCR versus virus isolation in chicken embryos and shows their diagnostic effectiveness in detection and subtyping of influenza A virus. The tests were evaluated with use of 185 specimens from humans, animals, and birds. IAVchip DNA microarray demonstrates higher diagnostic effectiveness (99.45%) in early influenza A diagnosis as compared to the real-time PCR (98.38%) and RT-PCR (96.22%), thus showing its clear superiority. Diagnostic sensitivity of IAVchip DNA microarray (100%) exceeds the same of RT-PCR (95.95%) and real-time RT-PCR (97.96%) in the range of estimated confidence intervals. IAVchip DNA microarray and real-time RT-PCR displayed equal diagnostic specificity (98.85%), while diagnostic specificity of RT-PCR was 96.40%. IAVchip DNA microarray has an advantage over the other tests for influenza A diagnosis and virus identification as a more rapid method that allows performing simultaneous detection and subtyping of about tens of specimens within one experiment during 8–10 hours. The developed IAVchip DNA microarray is a general test tool that enables identifying simultaneously 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A virus and also to screen the influenza A viruses from humans, animals, and birds by M and NP genes.

Owing to the high mutation rate of the surface HA and NA glycoproteins of IAV subtyping appears to be difficult; therefore, it is necessary to use supplementary methods and tools of diagnosis as well as their combinations to get reliable results [4,8]. High pathogenicity of influenza viruses and great economic and social damage caused by the infection requires rapid and correct diagnosing. The Scientific World Journal So far, the DNA microarray-based detection is one of promising techniques in differential diagnosis of IAV infection, since it allows combining efficiency of the nucleic acid amplification and ample capacities of screening with use of biochip [9][10][11][12][13][14].

Objectives
IAVchip DNA microarray [15,16] for early IAV detection and differentiation has been developed at the RIBSP (Research Institute for Biological Safety Problems) and now the ways of its use in practice are investigated. The objective of the study was to evaluate diagnostic effectiveness of IAVchip DNA microarray in differential detection of 16 hemagglutinin and 9 neuraminidase IAV subtypes as compared to virus isolation in chicken embryos, real-time PCR, and RT-PCR. Sampling was carried out following WHO recommendations [17]. Nasal smears (swabs) were taken from humans and horses using dry sterile probes with cotton tips; cloacal swabs were taken from birds in the same way. After sampling the cotton pellet (working part of the probe) was placed into a cryotube with sterile transport medium (transport media consisted of Hanks balanced salt solution supplemented with 10% glycerol, 200 U/mL penicillin, 200 mg/mL streptomycin, 100 U/mL polymyxin B sulphate, 250 mg/mL gentamicin, and 50 U/mL nystatin). Cryotubes with samples were transported in liquid nitrogen (−196 ∘ C). 3.3.2. Viral RNA Amplification. One-step RT-PCR was performed with use of Super Script III Platinum One-Step Quantitative RT-PCR System ("Invitrogen, " USA) following the manufacturer's instructions. The universal primer pair selected for the terminal highly conservative regions that are present in all IAV genome segments (MBTuni-12 and MBTuni-13) has been used in multisegment amplification [18]. Conditions of RT-PCR are as follows: SuperScriptIII RT/PlatinumR Taq Mix-1 L; 2X Reaction Mix-25 L; primers 10 M-1.4 L each; RNaseOUT-1 L; RNA (1 pg to 1 g)-10 L; DEPC-treated water up to 50 L [15]. Fluorescent labeling was made via direct integration of Cy5-dCTP ("DNA-Synthesis, " Russia) immediately in the process of RT-PCR and the reaction mixture contained extra 33 M of 1 mM Cy5-dCTP.

Assay with Use of IAVchip DNA
Microarray. An experimental batch of microarrays was printed on aldehyde substrate (Vantage Aldehyde Slides "CEL Associates") by the method of contact printing of oligonucleotide probes in Nano Print LM 60 ("Arrayit, " USA) in accordance with the scheme on Figure 1. The scheme includes the probes corresponding to hemagglutinins HA1-HA16 and then to neuraminidases NA1-NA9. In addition there is a universal oligonucleotide probe to the region of IAV genes that encode M and NP proteins shared by all IAV subtypes. Oligonucleotide probes used in the experiments are shown in Table 1.
One microarray slide allows simultaneous analysis of 16 different IAV strains and isolates.
IAVchip DNA microarray was validated with use of IAV reference strains of different subtypes, origin, and biological characteristics [16].

Hybridization.
To 1 L of PCR-mixture containing Cy5-cDNA hybridization solution was added; the total volume was brought with H 2 O up to 50 L and heated in the solid-state thermostat at 99 ∘ C for 2 min and then cooled in ice for 2 min and at once applied onto the microarray. In parallel the oligonucleotide probes on the microarray were denatured by boiling of the slide in H 2 O for 1 min followed by incubation in 96% ethanol (C −20 ∘ C) for 1 min. After that the slide was dried by centrifugation at 300 g for 2 min. Hybridization was performed with use of a frame for 16 subarrays FAST Frame ("Whatman", USA) for 2 h at 37 ∘ C and stirring at 250 rpm. After hybridization the slide was rinsed in 3×SSC buffer for 2 min and in 1×SSC buffer for 2 min to remove unbound molecules of the sample and hybridization buffer. After that the frame was removed and the slide was rinsed with water for 2 min. It was dried by centrifugation at 300 g for 2 min. The Scientific World Journal Scanning. The microarrays were scanned in InnoS-can710AL ("Innopsys, " France) with 5 m resolution. Fluorescent scanning was performed at wavelengths 532 nm and 635 nm. The resulted images were processed with use of the software Mapixver. 5.5.0 ("Innopsys", France).

Virus Isolation in Chicken Embryos.
Virus isolation was performed in 9-10-day chicken embryos (CE) following the standard procedures where influenza virus production was confirmed by identification in hemagglutination inhibition (HI) test [19].  [21,23], and for detection of X5N1 subtype according to WHO guidelines (2007) [22]. Specific regions of IAV cDNA were produced in GeneAmp PCR 9700, Applied Biosystems. Table 2 shows oligonucleotide primers that were used in the study.

Detection of Influenza A Virus in Clinical Specimens.
Diagnostic effectiveness of IAVchip DNA microarray in comparison to the virus isolation in chicken embryos, real-time RT-PCR, and RT-PCR for IAV detection and subtyping was evaluated by testing 185 clinical specimens from humans, animals, and birds.
"True" state of infection is determined by the most accurate diagnostic method that is called "gold standard. " In diagnostics and identification of the influenza infection virus isolation in chicken embryos followed by identification in HI is the "gold standard" [19]. Table 3 shows the results of detecting IAV in clinical specimens by various methods in comparison to virus isolation in chicken embryos.
In our experiments IAV was isolated by the method of virus isolation in chicken embryos from 98 (52.97%) specimens out of 185 clinical samples taken from humans, animals, and birds.

Comparison of Various Tests for Influenza A Diagnosis.
Sensitivity and specificity of each test were determined to evaluate effectiveness and reliability of influenza A diagnostic tests. Sensitivity and specificity indices, as well as positive and negative prognostic values of the developed IAVchip DNA microarray, of RT-PCR and real-time RT-PCR with 95% confidence interval are displayed in Table 4.
In influenza A diagnosis IAVchip DNA microarray and real-time PCR demonstrated equal diagnostic specificity (98.85%), while the same characteristic of RT-PCR was 96.40%. Diagnostic specificity of IAVchip DNA microarray and real-time PCR were in the range of the confidence intervals of the RT-PCR specificity. The limit of the 95% confidence intervals for RT-PCR was 93.70%-99.10%.
PPV and NPV values of IAVchip DNA microarray were 98.99% and 100%, respectively. PPV and NPV of the real-time RT-PCR were also in the range of the estimated confidence intervals.

Influenza A Virus Subtyping with Use of IAVchip DNA
Microarray. IAVchip DNA microarray, real-time RT-PCR, and RT-PCR were used to subtype IAV from 98 samples proved to be positive by the method of the virus isolation in chicken embryos. The IAV were subtyped by the methods of RT-PCR and real-time RT-PCR in 3 steps: identification of the virus (step 1), detection of HA (step 2), and NA (step 3). IAVchip DNA microarray was used to subtype simultaneously the tested IAV with probes specific to hemagglutinin 1-16 and neuraminidase 1-9 genes. Table 5 shows examples of IAV subtyping with the help of IAVchip DNA microarray in the form of histograms.
As Table 5 shows IAVchip DNA microarray makes possible simultaneous IAV subtyping in the tested samples. Possibility in principle to use the DNA microarray for diagnosis and subtyping of various influenza viruses has been demonstrated by examples of seasonal A/H1N1 and A/H3N2 viruses, equine influenza A/H3N8 virus, and highly pathogenic avian influenza A/H5N1 virus. In the assay of the samples the value of specific fluorescence reliably exceeded the value of the background fluorescence ( < 0.05).

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Discussion
IAV genome variability begets a very important diagnostic problem that consists in need of rapid and accurate method of diagnosis not only of existing but of emerging viruses for efficient influenza surveillance at the global level [25,26].
Biological microarrays are developed today in many countries. Biochips for diagnosis of the human influenza of subtypes H1N1, H3N2, including H5N1, are set forth [27]. It should be noted that various approaches were used. MChip was developed with use of probes only to M gene of the influenza virus, but further on it was supplemented with probes to hemagglutinin and neuraminidase genes (FluChip) [11]. The microarrays proposed by Huang et al. [1] and Teo et al. [28] contain probes to the hemagglutinin and nonstructural protein genes of the influenza B virus. These diagnostic microarrays can be used only for diagnosis of the seasonal influenza because reassortment and mutations so characteristic of the influenza virus result in emergence of new subtypes that are isolated both from animal and bird populations and from humans.
There are also microarrays for identification of all IAV subtypes by both hemagglutinin and neuraminidase genes. Xueqing et al. [29] used 52 oligonucleotide probes for typing IAV. The viral cDNA was amplified in multiplex PCR with 25 pairs of primers. Multiplex PCR for amplification of the influenza virus cDNA with use of 25 pairs of primers was performed by Han et al. [14]. The component structure was optimized for performance of the reaction in 4 tubes. Quan et al. [30] developed microarray GreeneChipResp that enables identifying 20 respiratory viral infections apart from typing all subtypes of IAV.
On the basis of the oligonucleotide microarray IAVchip that allows detecting and subtyping IAV has been developed at the RIBSP [15]. This microarray is universal for all subtypes of IAV and when necessary enables screening the IAV not only by HA and NA, but by M and NP genes as well.
Diagnostic effectiveness (DE) of the test expressed as percentage ratio of the number of true test results to the total number of findings for IAVchip DNA microarray was 99.45%, for the real-time PCR and RT-PCR; it was 98.38% and 96.22%, respectively.
The majority of authors propose in their papers using microarrays for the human influenza viruses [9,11,31], whereas our biological chip is designed to identify influenza viruses from humans animals and birds.
Analysis of these data shows that IAVchip DNA microarray for IAV diagnosis and identification has the advantage over the other tests in effectiveness and is more rapid owing to possibility to detect and subtype several influenza A viruses simultaneously in one experiment.
It should be noted that in the course of testing clinical specimens with use of IAVchip DNA microarray in one reaction both influenza A virus identification and subtyping of tens of viruses take place during 8-10 hours. Subtype information is especially important, for example, in the South-Eastern Asia, where subtypes A/H3N2, A/H1N1, and A/H5N1 can simultaneously circulate in a body.
So, possible usage of the microarray in clinical practice for influenza A diagnosis and subtyping of the viruses isolated both from humans and from animals and birds is shown.

Ethical Approval
Ethical approval was not required. Step 2 Step 3 Step 1 Step 2 Step 3 Step