The 2009 pandemic influenza A (H1N1 09), a major cause of health burden, has gained worldwide attention of scientists for better diagnosis and treatment. It is one of the important causes of acute respiratory infection and causes disease in any age group. However, the infection rate is higher among infants and children.1-4 It also accounts for other complications like pneumonia, viremia, myositis, myocardial dysfunction and encephalopathy.5 The H1N1 09 spread in 214 countries with more than 18306 deaths.6 Developing countries are comparatively at more risk because of lack of hygiene, overpopulation, poverty and scarcity of modern tools for the diagnosis and treatment. The influenza virus is known for continuous antigenic change through antigenic drift and shift.7 This property of the virus has a significant effect on prevention (vaccination), diagnosis and treatment. Although an emergence of new strain of the virus cannot be stopped, its spread from the origin might be limited by preventing country to country transmission. It has been reported that more than 90.0% of cases of H1N1 endemic occurred in England by in-country transmission.4,8 From the 2 pandemics (Severe Acute Respiratory Syndrome and H1N1 09) in the last decade, it is clear that rapid identification and proper surveillance tremendously help respond to the outbreak in short time, providing security to global public health.

One of the major preventions from circulating strains of influenza virus and the complications associated with the viral infection is by vaccination. As the virus is seasonal, the vaccine needs to be prepared according to the prediction that certain strain would circulate in a particular season.9 Also, antiviral agents against influenza are found to be effective when used early in the course of illness. There are four drugs available against influenza virus: amantadine, rimantidine, zanamivir and oseltamivir.10 Some studies have reported the resistance pattern of the H1N1 09 virus to amantadine and oseltamivir.11-13 Rapid detection of such resistance is clinically important in reducing the time between diagnosis and initiation of individualized therapy. Therefore, close monitoring of the prevalence of antiviral resistance and discoveries of drugs and potent vaccines are necessary to control influenza burden.

Another important aspect of controlling influenza burden is early diagnosis. However, diagnosis by a physician is difficult because many of the respiratory viruses produce similar symptoms like influenza. Therefore, clinical laboratory diagnosis is mandatory for correct and early diagnosis. Although several tests have been developed to detect influenza viruses, they have different sensitivity, specificity, throughput, cost, availability, and other factors involved in from pre-analytical to post-analytical stages of clinical laboratory testing. These factors play a crucial role in the selection of diagnostic test method in the clinical laboratory. Use of highly sensitive and specific test to yield accurate result in relatively short time is beneficial to patients by reducing the adverse effects and complications with the use of appropriate drugs and antiviral therapy on time.14

Every patient and clinician wish to know the causation of disease as early as possible after the illness begins. To meet this aim, rapid tests to detect influenza virus have evolved. In the past decade, several tests have been developed and the use of rapid tests is found to flourish day in day out. This is true because of their ability to provide test results within about half an hour with easy interpretability. Although these tests have not been able to gain trustworthiness because of several limitations like frequent false positivity and false negativity, they are known to yield better results with sample having higher titer, as in children than in adult cases.31 Especially in developing countries where the PCR technique is not available in primary health care settings, they aid in clinical decision making and have been found to be used solely for diagnostic purpose. These tests are handy and also known as near patient or point of care testing methods. Today's focus on rapid identification of influenza should be centered to increase the sensitivity and specificity of these tests, so that they can be of potential use in early and accurate diagnosis of influenza.

The commercially available rapid antigen tests are mostly immunoassays that target the antigen of influenza virus. However, they have relatively poor sensitivity in comparison to virus culture technique and PCR-based assays. In one study using QuickVue Influenza A+B (Quidel) with reference as RT-PCR, they found the sensitivity and specificity for the detection of H1N1 09 was 20.0% and 99.0%, respectively, suggesting the use of RT-PCR for the clinical diagnosis.29 Other studies have, however, found its sensitivities up to 51.0% and 53.3% in comparison to RT-PCR.32,33 In another study using two rapid antigen tests, BD Directigen EZ flu and TruFlu, Meridian, they got a better performance with former kit than the later one. The sensitivity and specificity for H1N1 09 detection with BD Directigen EZ flu was 20.6% and 99.0%, whereas those of TruFlu, Meridian were 9.7% and 98.2%, respectively.34 Tsao, et al.,35 recently analyzed four rapid tests, namely BinaxNow test, QuickVue test, TRU test, and Formosa Rapid test, and found their detection limit to be lower than that of RT-PCR. Nevertheless, they found the sensitivity of Formosa Rapid test to be similar to QuickVue test, relatively more sensitive than the other 2 kits for H1N1 09 diagnosis. Furthermore, another study using SD Bioline Influenza Antigen Test® kit found a poor performance, with sensitivity and specificity of 44.0% and 99.9%, respectively, for H1N1 09 diagnosis.36 In contrast, a recent study has revealed 90.0% sensitive Actim Influenza A&B kit (Medix Biochemica, Joensuu, Finland) for the detection of influenza A virus among 1-3 years old children.37 The test performed with QuickVue influenza test in 6 months to 14 years of children showed 67.5% sensitivity and 96.0% specificity for influenza viruses.38 This indicates that pediatric group have generally higher viral load fair enough for the kits to detect the virus in comparison to adults or other patients. The next important thing about the rapid kits is that their sensitivity and specificity are inconsistent.31

In comparison to PCR-based assays, direct fluorescence antibody assay (DFA) confers cheap and fast result, although it is not as sensitive as the PCR test. Several studies have been performed, especially surveillance ones, solely based on the DFA test.39 The technique involves the staining of cells from the sample, followed by bioconjugation of antibodies to the fluorescent dye. The time frame to obtain the results is between 2 and 4 h.40,41 The sensitivity of the DFA tests for influenza virus is found to be between 70.0% and 100.0%. Similarly, specificity ranges from 80.0% to 100.0%,22 indicating that the sensitivity is comparatively higher than that of rapid antigen detection kits and lower than the PCR tests. This means that the negative results need to be confirmed by the PCR-based assays. Although some studies recommend the use of DFA as well as SVC together, to increase the sensitivity of the test by 5.0-15.0% than the DFA alone, multiple virus infections cannot be ruled out.22 Recent study has shown that, particularly for H1N1, the sensitivity and specificity of DFA were 62.0% and 100.0%, respectively, when compared to RT-PCR, with variability in age. The performance of the test was better with children below 10 years of age, whereas the sensitivity of the test decreased with persons above 30 years of age.42 In contrast, the sensitivity of DFA was 93.0% with adult in a study by Pollock, et al.,41 in 2009 for H1N1 09. In another study, the sensitivity of DFA is shown to be lowered to 44.4% in comparison to RT-PCR for H1N1.43 Similarly, the study comparing DFA, rapid antigen test and RT-PCR showed that the former two tests have comparatively low sensitivity (38.7% and 18.2%, respectively) in comparison to RT-PCR, suggesting the confirmatory test as RT-PCR for H1N1 09.44

However, the DFA test requires skilled expertise for fluorescence microscopy. Furthermore, the DFA test is carried out in shift basis generally in the clinical settings, which prolongs the turnaround time. Thus, it does not seem to be a suitable test in regard to clinical laboratory diagnosis of influenza virus in comparison to PCR based assays.

Number of studies have reported that PCR-based assays overcome several demerits possessed by other techniques such as rapid influenza detection tests (RIDTs) and SVC with respect to sensitivity, specificity and time frame.45,46 For both qualitative and quantitative approach of diagnosis, the RT-PCR is regarded as the king of diagnosis for the viruses, including influenza virus. There are 2 types of RT-PCR. One step RT-PCR involves the use of oligo-dT or random primers for reverse transcription. Next is two step RT-PCR where reverse transcription is performed and then PCR is carried out. Briefly, RNA extracted from the influenza sample is purified and transcribed using the oligonucleotides specific to the target sequence, producing cDNA.47

The first PCR-based assay in detecting influenza virus was described in 1991 by Zhang and Evans.48 Since then, many modifications and highly sensitive methods based on PCR have been developed. For instance, PCR-enzyme immunoassay has been found to have better performance than the culture method in cases with 5-9 days of illness.49 Similarly, RT-PCR followed by electrospray ionization mass spectrometry is able to detect the culture-unidentified viruses.50 When the H1N1 outbreak occurred in 2009, CDC recommended the use of one step RT-PCR technique targeting the matrix gene of H1N1 09.51 The type and subtype specific detection methodology using this technique have been already described using the hemagglutinin gene as target in a single tube, which minimized the cost and time required to analyze the sample for each type and subtype of the influenza virus.49 Other PCR-based assays such as ResPlex II (Qiagen, Hilden, Germany), MultiCode PLx RVP (EraGen Biosciences, Madison, WI, USA), Seeplex RV (Seegene Inc., Seoul, Korea), NGEN RVA ASR (Nanogen Inc., San Diego, CA, USA), xTAG RVP (Luminex Molecular Diagnostics, Toronto, Ontario, Canada), ProFLu+ (Prodesse Inc., San Diego, CA, USA) and ProFLu-1 (Prodesse Inc., San Diego, CA, USA) are also available.22 Among them, the later three are approved by Food and Drug Administration (FDA) for the detection of respiratory viruses, including influenza viruses. These multiplex RT-PCR techniques are highly sensitive and specific to give result in comparatively less time than the viral culture and EIA techniques.

Considering the difference between PCR-based assays and other techniques such as RIDTs and SVC in the aspect of merits of diagnosis of influenza virus, PCR method seems to be effective. However, intensive research in medical diagnosis has led even more effective diagnostic tools, based on PCR assays. For instance, nucleic acid sequence-based amplification tests are known to be more sensitive than the RT-PCR tests for the detection of influenza virus. In this technique, three enzymes are used, namely AMV-RT, T7 RNA polymerase and RNaseH. It can detect the H1N1 09 in 90 min with sensitivity and specificity of 100.0%.52 Moreover, multiplex nucleic acid-based amplification test along with an enzyme-linked oligonucleotide capture has shown to rapidly and specifically detect a single virus, including influenza viruses A and B from a multiplexed group, reducing laboratory testing time and enabling high throughput screening.53

Although nucleic acid-based amplification assays are found to yield better sensitivity in comparison to other methods, more other techniques are being studied by FDA. For instance, Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+) (Nanosphere, Chicago, IL, USA), which is recently cleared by FDA, is an automated method for H1N1 09 diagnosis with the sensitivity and specificity of 99.5% and 100.0%, respectively.54 Accordingly, Cepheid Company (Sunnyvale, CA, USA) developed a Flu A (Influenza virus) Panel test which is an FDA-cleared fully automated technique for the detection in 45 min.55

The knowledge on the genetic basis of resistance to drugs has provided molecular techniques to quickly detect the mutations conferring resistance to the drugs. For instance, one of the most widely used drugs against influenza infection, oseltamivir resistance, arises due to histidine to tyrosine substitution in the neuraminidase active site (H275Y).56 The techniques available to monitor the drug resistance include neuraminidase inhibition assay, pyrosequencing, use of genotype specific primers, or real time RT-PCR,56 all of which have been known to be time consuming. Sequencing methods such as pyrosequencing provide detailed genetic information about the resistance pattern and also estimate relative proportion of variant and wild-type viruses in a mixed population sample and can be applied directly in clinical setting.57 However, they need additional steps after initial PCR, specialized person to operate the sequencing instruments and the softwares.24 Real-time quantitative RT-PCR provides a rapid, highly sensitive, and specific alternative to sequencing.24 However, it is either specific for seasonal influenza A or needs multiple primers to detect H1N1 09.56 RT-PCR, combined with pyrogram techniques, has provided the detection of H275Y sequences in H1N1 09 within 8 h with sensitivity similar to pyrosequencing method for seasonal H1N1.56 Similarly, cycling probe real-time PCR assay has been shown to provide test results within 3 h after receiving the samples for resistance to oseltamivir and also to be able to detect both seasonal as well as H1N1 09 viruses resistant to oseltamivir.58 However, the major drawback is that new sets of primers and probes have to be developed in the event when a novel drug-resistant strain would emerge during treatment.58

Influenza virus is a significant global health burden. Timely diagnosis and initiation of treatment reduce the stigma of the disease, and also helps in outbreak control. Sole interpretation by a clinician, based on the symptoms presented by a patient, is not specific, as the virus has symptoms common to other known respiratory viruses. Therefore, clinicians are always in need of a confirmatory test for the influenza infection as soon as the illness initiates. The culture methods are time-consuming and are not as sensitive and specific as to make clinicians to start with antiviral therapy. However, they are still being used in many laboratories, suggesting that the influenza diagnosis by culture methods needs substantial quality check and continuous monitoring to increase the quality of report. The fast and specific RIDTs, also called as point of care tests, are also not appropriate for the diagnosis of influenza virus because of their poor performance. However, they are simple, cheap, easily interpretable and handy. If they had been showing similar or better performance than that of PCR-based assays, they would have been the major technique to detect the influenza virus. However, they too need to be studied well and improved to the extent where they can be used for accurate and specific diagnosis of the virus.

Recent advancement in the PCR-based assays have become popular in advanced countries. However, we cannot deny the fact that these popular models also have several limitations, ranging from nucleic acid extraction to result interpretation. Even a negligible amount of error has potential effect on the result interpretation of the highly sensitive tests. Therefore, they have also higher possibilities of yielding false positive results in comparison to viral culture methods. Thus, validation of reagents, application of proper positive and negative controls, and use of automation could reduce the chances of errors in molecular diagnosis of influenza infection. On the other hand, cell culture methods and RIDTs should undergo standardization, quality assurance and quality control measures in order to validate the results. Hence, further studies are needed to validate the interpretation of influenza virus diagnostic techniques. Besides many merits and demerits, PCR based-assays are so far suggested technique for the diagnosis of influenza virus. Therefore, clinicians should rely on the RT-PCR test results of patient and correlate with the clinical aspects of the patient.