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Abstract

Objective:

To investigate the detection of influenza viruses by three different methods.

Methods:

Nasopharyngeal swabs were collected from patients with influenza symptoms and examined for influenza A and B viruses using a rapid antigen test, a multiplex polymerase chain reaction (PCR) test and a shell vial cell culture test.

Results:

Using the shell vial cell culture test, the rapid antigen test and the multiplex PCR test in 130 patients, 31 (23.8%), 24 (18.5%) and 24 (18.5%) samples, respectively, were positive for influenza A and 10 (7.7%), nine (6.9%) and four (3.1%) samples, respectively, were positive for influenza B. Compared with the shell vial test, the sensitivity, specificity, and positive and negative predictive values of the rapid antigen test were 77.4%, 93.3%, 80.0% and 93.1%, respectively, for influenza A, and 90.0%, 95.8%, 64.2% and 99.1%, respectively, for influenza B. The corresponding values for the multiplex PCR test were 77.4%, 95.9%, 85.7% and 93.1%, respectively, for influenza A, and 40.0%, 97.5%, 57.1% and 95.1%, respectively, for influenza B.

Conclusions:

The multiplex PCR test and the rapid antigen test are both effective in the detection of influenza A and B viruses.

Keywords

Antigen Influenza Influenza A virus Influenza B virus Multiplex polymerase chain reaction test Rapid antigen test Shell vial cell culture

Introduction

Influenza A and B viruses cause respiratory infections, leading to symptoms such as fever, sore throat, cough, myalgia and headache. Both viruses are highly infectious and can spread easily within the population.¹ Influenza epidemics and pandemics can be associated with high morbidity and mortality; elderly people with chronic diseases, children and immunosuppressed patients are particularly at risk.^2,3

Currently available antigen tests have a relatively low sensitivity that limits their use in the diagnosis of influenza.⁴ Polymerase chain reaction (PCR) detection methods are generally more sensitive than antigen tests and can sometimes distinguish between strains such as H1N1, H3N2 and H5N1, in addition to detecting the presence of influenza virus.⁵

In recent years, the laboratory identification of influenza viruses has increased in importance worldwide due to influenza epidemics and pandemics, especially those relating to influenza H1N1. In the present study, the sensitivity and specificity of a rapid antigen test and a multiplex PCR test for the detection of influenza A and B viruses were compared with the results of shell vial cell culture, as cell culture is considered the gold standard test for the identification of influenza viruses.

Patients and methods

Patients

Patients with influenza-like symptoms including sore throat, runny nose, cough and fever, who were attending the Chest Diseases, Paediatric or Infectious Diseases Clinics of Meram Faculty of Medicine, Necmettin Erbakan University, Konya, Turkey, or four primary health care centres in Konya, Turkey, during the 2008 – 2009 and 2009 – 2010 autumn/winter seasons (between October and March) were recruited sequentially to the study. Patients who had experienced influenza-like symptoms for > 4 days and those with immunosuppression were excluded from the study. Each patient's age, gender and symptoms were recorded. Patients were classified as children (< 18 years of age) or adults (≥ 18 years of age).

Written informed consent was obtained from all patients or their parents/guardians. The study protocol was approved by the Ethics Committee of Meram Faculty of Medicine, Selcuk University, Konya, Turkey (No. 2007/259).

Respiratory Samples

Two nasal or throat swabs were taken from each patient by physicians working at the health care centres or hospital. One swab sample was used for the rapid antigen detection test, which was performed at the health care centre or hospital. The other swab sample was placed in viral transport medium appropriate for the isolation of influenza virus (Vircell, Santa Fé, Spain), refrigerated and then transported to the Central Microbiology Laboratory at Meram Faculty of Medicine, for use in the shell vial cell culture test and the multiplex PCR test. Samples that could not be sent immediately were kept at 4 °C for ≤ 5 days and were then preserved at -70 °C. Prior to analysis, samples were thawed at room temperature and mixed using a vortex to free and homogenize the swab content. The swab was then rotated and pressed against the side of the tube. The liquid content was poured off into a centrifuge tube in a sterile environment, and mixed at 1344 g for 10 min. The content was then divided into two tubes: one to be used for cell culture inoculation in the shell vial test and the other to be used in the multiplex PCR test.

Rapid Antigen Test

Rapid antigen detection was performed using the SD Bioline Influenza Antigen Rapid Test kit (Standard Diagnostics, Kyonggi-do, Korea) according to the manufacturer's instructions. Appearance of the control band, together with a test band corresponding to A or B antigen, was regarded as a positive result. Results from the rapid antigen test were available within 10 min.

Shell Vial Cell Culture Test

The rapid cell culture test was performed using the MDCK Shell Vials Test (Vircell), which is based on Madin–Darby canine kidney (MDCK) cells, according to the manufacturer's instructions. Detection of viral replication was by indirect fluorescent assay (IFA). All procedures were performed inside a sterile laminar flow cabin. Results from the shell vial cell culture test were available within 48 h.

Multiplex PCR Test

Multiplex PCR was performed using the Seeplex^® RV15 ACE detection kit (Seegene, Seoul, Korea) according to the manufacturer's instructions. All procedures were performed inside a sterile laminar flow cabin. Results from the multiplex PCR test were available within 5 – 6 h.

Statistical Analyses

The sensitivity, specificity and positive and negative predictive values for the rapid antigen and multiplex PCR tests were calculated using the shell vial cell culture test results as the reference values. All statistical calculations were made using SPSS^® software version 13.0 (SPSS Inc., Chicago, IL, USA).

Results

A total of 130 patients with symptoms of influenza were included in the study: 40 were recruited in the 2008 – 2009 autumn/winter season and 90 in the 2009 – 2010 autumn/winter season. The mean ± SD age of the patients was 29 ± 5.2 (range 0 – 65) years. The age categories, gender profiles and symptoms for the 130 patients are given in Table 1.

Table 1:

Characteristics of 130 patients with influenza-like symptoms

Characteristic	n	Fever	Sore throat	Congestion/runny nose	Cough
Boys	41 (31.5)	27 (20.8)	16 (12.3)	28 (21.5)	30 (23.1)
Girls	37 (28.5)	25 (19.2)	15 (11.5)	24 (18.5)	33 (25.4)
Men	21 (16.2)	6 (4.6)	10 (7.7)	11 (8.5)	3 (2.3)
Women	31 (23.8)	13 (10.0)	14 (10.8)	16 (12.3)	6 (4.6)
Total	130 (100.0)	71 (54.6)	55 (42.3)	79 (60.8)	72 (55.4)

Data presented as n (%) of patients.

The numbers of samples positive for influenza A or influenza B virus detected using the three methods are given in Table 2. The appearances of samples positive for influenza A or influenza B viruses using the shell vial cell culture method are shown in Figs 1 and 2, respectively.

Figure 1:

Appearance of a respiratory swab sample positive for influenza A virus using the shell vial cell culture method; viral replication was detected by indirect fluorescent assay

Figure 2:

Appearance of a respiratory swab sample positive for influenza B virus using the shell vial cell culture method; viral replication was detected by indirect fluorescent assay

Table 2:

Positive results for influenza A and influenza B viruses obtained from respiratory swab samples using three different detection methods in 130 patients with influenza-like symptoms

Parameter	Shell vial cell culture test	Rapid antigen test	Multiplex PCR test
Influenza A
Boys	15 (11.5)	13 (10.0)	9 (6.9)
Girls	12 (9.2)	9 (6.9)	10 (7.7)
Men	1 (0.8)	0 (0.0)	2 (1.5)
Women	3 (2.3)	2 (1.5)	3 (2.3)
Total	31 (23.8)	24 (18.5)	24 (18.5)
Influenza B
Boys	3 (2.3)	3 (2.3)	2 (1.5)
Girls	5 (3.8)	5 (3.8)	2 (1.5)
Men	1 (0.8)	0 (0.0)	0 (0.0)
Women	1 (0.8)	1 (0.8)	0 (0.0)
Total	10 (7.7)	9 (6.9)	4 (3.1)

Data presented as n (%) of patients.

PCR, polymerase chain reaction.

The sensitivity, specificity, and positive and negative predictive values of the rapid antigen test and multiplex PCR test, using the shell vial cell culture test results as the reference values, are given in Table 3.

Table 3:

Diagnostic values of a rapid antigen test and multiplex polymerase chain reaction (PCR) test for the detection of influenza A and influenza B viruses, using shell vial cell culture test results as the reference values, in 130 patients with influenza-like symptoms

Diagnostic value	Rapid antigen test		Multiplex PCR test
Diagnostic value	Influenza A	Influenza B	Influenza A	Influenza B
Sensitivity, %	77.4	90.0	77.4	40.0
Specificity, %	93.4	95.8	95.9	97.5
Positive predictive value, %	80.0	64.2	85.7	57.1
Negative predictive value, %	93.1	99.1	93.1	95.1

Discussion

The diagnosis of influenza virus infections is important as they can lead to epidemic and pandemic outbreaks, affecting a substantial proportion of the population and even causing death. In order to prevent the spread of the disease and to treat patients appropriately, influenza needs to be detected rapidly. A number of commercial rapid tests have been developed that can determine the existence of influenza A and B viruses within 10 – 30 min.⁶ The gold standard for viral diagnosis is the cell culture method; as in the present study, this method has been used as the reference point for other viral detection techniques in a number of other studies.^{6 – 10}

In the present study, results produced by the SD Bioline Influenza Antigen Rapid Test were compared with those from a shell vial cell culture method. Using the rapid antigen test, 24 (18.5%) samples were positive for influenza A and nine (6.9%) samples were positive for influenza B, compared with 31 (23.8%) and 10 (7.7%) positive samples for influenza A and B, respectively, detected using shell vial cell culture. This gave a sensitivity, specificity, positive predictive value and negative predictive value of 77.4%, 93.4%, 80.0% and 93.1%, respectively, for influenza A, and 90.0%, 95.8%, 64.2% and 99.1%, respectively, for influenza B. In a similar study of two rapid antigen tests carried out in 2003 by Dunn et al.,¹¹ the sensitivity and specificity were found to be 54.8% and 100.0%, respectively, for influenza A, and 62.5% or 56.3%, and 99.6%, respectively, for influenza B. As the pandemic influenza A H1N1 virus in 2009 was different to previous influenza A types, sensitivity for this virus using previously produced influenza rapid test kits ranged from 10 – 69% and the presence of low-titre influenza could not be determined.¹² In response, Chen et al.¹³ developed a rapid diagnostic kit using monoclonal antibodies against two nucleoproteins specific to the 2009 virus, and produced a sensitivity of 88.5% and specificity of 99.3% in patient samples. Thus, the sensitivity and specificity values of rapid antigen tests can vary widely.

As the currently available rapid tests that detect antigens have relatively low sensitivities, there remains a need for rapid tests that have both high sensitivity and specificity. Various molecular methods have been developed for the detection of influenza viruses, most of which are based on PCR techniques.¹⁴ The multiplex PCR test used in the present study is one such method and is able to detect more than one viral respiratory tract infection agent at the same time. In the present study, results produced by the multiplex PCR test were compared with those from shell vial cell culture. Using multiplex PCR, 24 (18.5%) samples were positive for influenza A and four (3.1%) samples were positive for influenza B, compared with 31 (23.8%) and 10 (7.7%) positive samples for influenza A and B, respectively, detected using shell vial cell culture. This gave a sensitivity, specificity, positive predictive value and negative predictive value of 77.4%, 95.9%, 85.7% and 93.1%, respectively, for influenza A, and 40.0%, 97.5%, 57.1% and 95.1%, respectively, for influenza B. Although the sensitivity and positive predictive value of multiplex PCR for influenza B were low, the other parameters were high, therefore the diagnostic value of this test is good enough for it to be used routinely.

Several studies have investigated the use of various molecular techniques such as agarose gel analysis, probe hybridization, nested PCR, multiplex PCR and PCR– enzyme-linked immunosorbent assay (ELISA) in the diagnosis of influenza viral infection. In most of these studies, the sensitivity and specificity of these techniques were calculated by comparing them with each other, and with standard methods such as isolation in cell culture.^9,10 In some studies investigating cell culture and PCR methods, the cell culture isolation rate was found to be less sensitive than PCR.^15,16 Delays and noncompliance with cold chain practice when sending samples to the laboratory, together with samples not being taken within 3 days of symptom onset, can affect the levels of infectious particles in clinical samples^17,18 and can therefore decrease the rate of virus isolation using cell culture. In the present study, commercial viral transport medium was used for the samples and cold chain conditions were adhered to during transport to the laboratory. A total of 31 samples positive for influenza A virus and 10 samples positive for influenza B virus were detected using the shell vial culture system; however, if there were factors inhibiting viral production, the actual number of positive samples may be higher.

With many influenza viruses, cytopathogenic effects are not substantial or do not occur at all. Therefore, in most influenza virus cell culture studies, haemagglutination or haemadsorption methods are used, together with observation of cytopathogenic effects. In cultures in which the existence of virus is determined using these methods, IFA and ELISA are used for direct detection and identification of the virus.¹⁹ In the present study, influenza A and B viruses were detected and identified using IFA after shell vial culture.

Influenza is widely seen in autumn and winter in Turkey, and can lead to epidemics. In the present study, samples were collected over two consecutive autumn/winter seasons. Detection of influenza A and B viruses by a rapid antigen test and a multiplex PCR molecular method were compared with the results of a commercial cell culture system. It was shown that both the multiplex PCR technique and the rapid antigen test are effective in the detection of influenza A and B viruses.

The shell vial culture system can produce results within 48 h. However, in order to assess the viral antigenic features and to isolate and detect influenza viruses using cell culture techniques with optimal sensitivity, samples should be taken under appropriate conditions. Transportation of samples, choice of cell for culture, application of various processes to obtain a high titre of viruses and viral identification are all important steps that may affect the accuracy of detection. The multiplex PCR test also requires careful transportation and preservation of samples in order to perform replication of the viral RNA. However, since viral culture is not required for this method, there is no need to preserve live viruses. The rapid antigen test can be performed at the location where samples are taken, so there is no requirement for sample transportation or preservation.

The correct diagnosis of influenza is important for the prevention of unnecessary antibiotic use in viral infections. Clinical and reference laboratories must choose the appropriate laboratory method for viral detection and identification, according to the available facilities and the speed with which the result is required. Molecular diagnostic methods require specialized personnel and so may be limited to tertiary health centres and reference laboratories, whereas the rapid antigen test can be performed in primary or secondary health care centres.

In this study, the rapid antigen test and multiplex PCR test gave similar results. A rapid antigen test containing antigen against nucleoproteins of epidemic and pandemic strains may be useful for the routine diagnosis of influenza in primary and secondary health care centres. The multiplex PCR test was the more accurate detection method, and may be used when diagnostic laboratory facilities, with suitably qualified personnel, are available.

Footnotes

Acknowledgement

This study was supported by a scientific research fund of Selçuk University School of Medicine, Konya, Turkey (No. 08102002).

Conflicts of interest: The authors had no conflicts of interest to declare in relation to this article.

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Comparison of the Detection of Influenza A and B Viruses by Different Methods