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Emerging known and unknown influenza viruses create profound threats to public health; platforms for rapid detection and characterization of influenza viruses are critically needed to prevent and respond to any potential outbreaks in Taiwan. During the past decade, quickly developing and far-reaching technology of biotechnology were wildly applied; the most commonly used methods to identify and quantify influenza infection at Taiwan CDC can be subdivided into three broader phases, phase 1, pre-SARS, and techniques measuring viral infectivity (viral plaque assay, TCID50, and immunofluorescence assay); traditionally cell culture based on virus isolation has been regarded as “golden standard” for the detection and diagnosis of virus infection, and it is the technique to which all other test methods have been compared. After SARS outbreak, the laboratory diagnostic technics were moved to the next phase, which are to examine viral nucleic acid and protein (qPCR, immunoblotting, ELISA, and hemagglutination assay). The development of molecular methods for the direct identification of specific viral genome from clinical sample is one of the greatest achievements during this period. The advance and current phase of the Center for Research, Diagnostics and Vaccine Development of Taiwan CDC is to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome (DNA microarray, liquid chip array, and SNP). Clearly nucleic acid amplification techniques including PCR, nucleic acid sequence-based amplification, and multiplex detection system are proven technologies for rapid detection and molecular identification for most known human influenza viruses. Influenza viruses are traditionally detected using specific antibody-based immunoassays or immune-fluorescence assays. On the other hand, RT-PCR and real-time RT-PCR assays using specific primers against vial nucleic acids were more advanced and specific and provide faster results than end-point assays of influenza detection and in many cases have sensitivities equal to or better than cell culture. Unfortunately, high mutation rates of influenza virus may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use irrelevant; therefore, it is highly demanded to develop rapid and universal identification and detection technologies. Furthermore, facing the growing threat of interspecies transmission of influenza viruses resulting in the emergence of new infectious pathogens, the DNA microarray was applied in Taiwan CDC for diagnosis, identification, sequencing, and subtyping of influenza virus. Recently, a new molecular biology-based microbial detection method for rapid identification of multiple virus types in one specimen has been developed. Microbial Detection Array (MDA) detects viruses using probes against genomic DNA sequence within 24 hours. Each probe tests for a particular sequence of DNA and small groups of probes can be used to check for specific viruses up to the species level, different from current PCR technologies that focus on small, prioritized sets of high-risk biological pathogens. MDA, however, can identify a broad range of organisms, including pathogens on a priority screening list, sequenced bacteria, or viruses that might not be anticipated. Such technology has great potential for improving diagnostic processes and in different applications. New technologies have been continuously added in the field of biomedical sciences, which has gradually enriched science and enormously improved the quality and quantity of research output and will provide better preparedness for the next outbreak from a new emerging influenza virus.
RIDTs are antigen-based tests developed for rapid diagnosis of influenza virus infections in POC settings. These tests use monoclonal antibodies that target the viral nucleoprotein and employ either enzyme immunoassay or immunochromatographic (lateral flow) techniques. Available in dipstick, cassette, or card formats, RIDTs can be completed in less than 30 min, with the results observed visually based on a color change or other optical signals. Due to simplicity in their use and the speed of obtaining assay results, RIDTs are commonly used for the diagnosis of influenza infections.
Several FDA-approved RIDTs are currently available on the market. Most of these tests can either detect or distinguish influenza A and B viruses, detect only influenza A viruses, or both influenza A and B viruses (but cannot discriminate influenza A and B). However, none of the RIDTs can distinguish between the different influenza A subtypes. Performance of RIDTs is dependent on the prevalence of circulating influenza viruses in the population. During peak influenza activity, positive predictive values are high and false positives are, therefore, likely to be observed. However, during low influenza prevalence, negative predictive values are high, with low positive predictive values. RIDTs have generally demonstrated high specificities (95%–99%) for the detection of seasonal influenza virus infections.
For diagnosis of seasonal influenza infections, RIDTs have demonstrated variable assay performance with sensitivities ranging between 10%–70%, with up to 90% specificity compared to standard RT-PCR-based assays. Performance of RIDTs have been shown to be better in children compared with adults (approximately 13% higher), potentially due to higher viral loads and longer viral shedding in children compared with adults. A meta-analysis of 159 studies involving 26 commercial RIDTs showed a sensitivity of 62.3% compared to RT-PCR approaches for diagnosis of influenza infections (both seasonal and pH1N1 virus infections). In this study, the performance of RIDTs for the detection of influenza A viruses was higher compared to influenza B viruses (percentage sensitivities were influenza A: 64.6% and influenza B: 52.2%).
During the 2009 H1N1 pandemic, RIDTs demonstrated sensitivities ranging between 10%–70% compared with RT-PCR-based assays. Using the BinaxNOW rapid antigen-based assay (Inverness Medical, Cologne, Germany), Drexler et al. reported an assay sensitivity of 11.1% while testing 144 PCR-positive clinical samples from Bonn, Germany. Early during the pandemic, a large study from New York reported 9.6% and 40% sensitivities using RIDTs BinaxNOW Influenza A&B test (BinaxNOW), 3M Rapid Detection Flu A+B test (3MA+B) compared to R-Mix culture. The low sensitivity of RIDTs during the pandemic could be attributed to poor sample quality and inexperience of the lab workers. A Centers for Disease Control and Prevention (CDC) study evaluating the performance of three different RIDTs (BinaxNOW Influenza A&B, Directigen EZ Flu A+B, and QuickVue Influenza A+B) for the detection of the pH1N1 virus had reported assay sensitivities of 40% for BinaxNOW Influenza A&B, 49% for Directigen EZ Flu A+B, and 69% for QuickVue Influenza A+B compared to a RT-PCR-based assay. Due to a high rate of false negatives, the CDC advised physicians not to discontinue antiviral therapy despite negative RIDT results. In another study, the QuickVue Influenza RIDT assay (Quidel, San Diego, CA, USA) showed an assay sensitivity of 51% in comparison with a PCR assay. Another study compared a RIDT QuickVue Influenza test with the RT-PCR-based assay and reported an assay sensitivity of 66% with 84% specificity. The positive and negative predictive values in this study were 84% and 64%, respectively. A study comparing RIDT and cell culture approaches with a multiplex respiratory viral assay (Luminex xTAG), reported a combined assay sensitivity of 17.8% for the Binax NOW Influenza A+B (Inverness, Scarborough, ME) and the 3M Rapid Detection Flu A+B (3M Medical Diagnostics, St. Paul, MN, USA). While testing nasopharyngeal aspirates from 970 young children, one study reported 84.1% sensitivity using a RIDT, QuickVue Influenza A+B ICT test (Quidel Corp., San Diego, CA, USA) compared with a viral isolation method.
For detection of avian influenza A viruses, RIDTs have demonstrated lower sensitivity compared RT-PCR-based approaches. The FDA recently approved AV Avantage A/H5N1 Flu RIDT developed by Arbor Vita Corporation, (Sunnyvale, CA, USA) for H5N1 detection. This test uses a combination of monoclonal antibodies and PDZ (Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), Zonula occludens-1 protein (zo-1)) domain containing recombinant proteins to detect NS1 protein from throat swabs and can be completed in 45 min Although RIDT have demonstrated variable sensitivity, they still remain the test of choice in most clinical virology laboratories around the world due to the speed in obtaining results, simplicity in assay procedure, and cost.
If study participants experienced symptoms of ILI, they performed a nasal swab to be tested for 22 viral targets (Additional file 1: Table S1) as described. Swab processing and PCR are described in detail in the supplementary data section (Additional file 1: Text Material S1). This step was taken to link ILI symptoms with the detection of defined viral pathogens. In addition, if the PCR tested positive for H1N1, a nurse visited the participant 14 days (on average) after onset of symptoms in order to obtain a blood sample, which allowed study of the humoral and cellular immune response early after infection. This was also performed for participants infected with coronavirus as a control group.
Sera, collected at study entry and after the flu season in the spring of 2010, and during a home visit in case of a positive H1N1 or coronavirus PCR (detected from a nasal swab), were stored at −80°C until testing. Single radial haemolysis (SRH) test against A/H1N1/California/07/2009 was performed at the Department of Physiopathology, Experimental Medicine and Public Health of the University of Siena according to procedures described in detail elsewhere. Diameters of the haemolysis area for each serum tested were measured. Sera with areas of haemolysis equal to or higher than 4 mm2 but lower than 25 mm2 were considered to indicate seropositivity but not protection; haemolysis areas equal to or higher than 25 mm2 were considered to indicate seroprotection.
The VN assay is another technique used to measure induction of virus-specific antibodies following natural infection or vaccination, and is routinely used for the detection of antibody titers of either seasonal or avian influenza A virus strains. This approach is based on the ability of virus-specific antibodies to neutralize virus, thereby preventing viral infection of cells. The reciprocal of the highest serum dilution at which virus infection is completely blocked is considered the virus neutralization titer. Although the VN assay is more sensitive compared to the HAI assay, its application for routine diagnostic application is restricted due to the need of use of infectious viruses in certified BSL2+ and BSL3 laboratories.
Among the 279 patients tested by RT-PCR, 69 (24.7%) had influenza A virus and 13 (4.7%) had other respiratory viruses detected. Complete information concerning clinical presentation and routine examination was available for 190 patients (68.1% of the total), 61 of the FLU-A-positive cases and 129 of the FLU-A-negative cases. Data on the number of samples, clinical and univariate characteristics, P value and odds ratio (OR) (as well as the 95% confidence intervals (CIs)) in FLU-A-positive and in FLU-A-negative cases can be seen in Table 2. In comparison to FLU-A-negative ILI-case patients, cough was more likely to be reported in patients whose RT-PCR tested positive for influenza A virus (Table 2). Patients infected with FLU-A experienced more cough than patients infected with other viruses or those viruses free (χ2, p<0.001). Other clinical characteristics (such as sore throat, headache) appeared to be more frequent in those infected with the FLU-A virus than those non-infected, but the difference was not statistically significant. FLU-A-infected patients presented with a mean temperature of 38.6°C [95% CI (38.4, 38.7)] which on average, commenced 1.3 days before admission [IQR 1–2]. Approximately 30% of FLU-A-infected patients reported hyperpyrexia (temperature >39°C). The median age of patients who tested positive for influenza A was 32 years [IQR 27–47], and 44.3% were male. There was no significant difference in male and female rates. There was no difference in the neutrophil percentage between those whose RT-PCR tested positive for influenza A and those who tested negative. The clinical and univariate characteristics of influenza-A virus ILI-case patients are showed in Table 2.
For RT-PCR-positive patients, we undertook telephone follow-up to collect further data (Table 1). In 20 FLU-A-positive cases, 4 (20%) received antiviral treatment within three days of the onset of symptoms. A further 21.2% (11 of 52) of these RT-PCR-positive patients had been vaccinated. A further 11.3% (8 of 71) reported chronic comorbid conditions (diabetes mellitus in four and hypertension in four). The median duration of fever was four days [IQR 3–5]. The median time for disappearance or abated of systemic symptoms in the presence of influenza virus infection, or other viral infections was 4 days [IQR 3–7] and 3 days [IQR 2–6] respectively.
Individual respiratory clinical samples were collected. These included throat swab, nasopharyngeal swab, bronchoalveolar lavage, and lung biopsy according to each patient’s condition. Throat and nasopharyngeal swabs were collected using a rayon or Dacron hyssop in a plastic tube containing 2–3 mL viral transport medium. Bronchoalveolar wash and lung biopsy samples were collected by trained medical staff and placed in plastic bottles containing 15–20 mL viral transport medium. All samples were kept at 2°C–8°C after sampling and during transport to a local molecular biology laboratory and stored at −80°C until testing.17
A total of 88 study patients with confirmed H1N1 by quantitative qRT-PCR from six health districts of Oaxaca in Mexico were examined for other microbial infections. These patients were recruited from April 2009 throughout December 2012. The present study meets the operational definition criteria for ILI cases recommended by the World Health Organization.45
The Taiwan National Influenza Center (Taiwan NIC) was established in Taipei on July 5, 2006, a special date to be chosen to remember that Taiwan was removed from the WHO's list of areas that had been affected by SARS for three years. The WHO Global Influenza Surveillance Network was established in 1952. It comprises 5 WHO collaborating centers (WHO CCs) on influenza and 112 institutions in 83 countries, which are recognized by WHO as WHO National Influenza Centers (NICs). Although Taiwan NIC has not been recognized as a member of the WHO's network of influenza centers, Taiwan has regularly volunteered to send Taiwanese influenza isolates to the WHO CCs since 1979, making it part of the international flu virus supervision network.
During the post-SARS period, Taiwan CDC speeds up and strengthens domestic research on influenza and expands the exchange of information with our international counterparts, because Taiwan would face a great threat if another SARS epidemic and flu outbreak overlapped. Given the mutations of avian flu viruses, Taiwan CDC must fully equip itself to face any possible epidemic or pandemic. Thus, laboratory diagnostic technologies were established during this period, including multiplex RT-PCR/PCR, multiplex suspension beads array, microarray, loop-mediated isothermal amplification (LAMP), and high throughput sequencing. The diagnostic capacity of Taiwan CDC would be able to handle suspected clinical specimen around 1,500 cases in a day at maximum.
Among the 279 swabs collected, 82(29.4%) samples tested positive for at least one respiratory virus by multiplex RT-PCT. The temporal distribution of viral agents from confirmed cases tested by RT-PCR is shown in Figure 1. Influenza viruses (FLU) were the predominant viral etiology comprising 80.5% (23.7% of the total specimens) of the confirmed cases between June 2010 and May 2011 in Beijing. Almost all of these FLU-positive specimens were FLU-A cases, which accounted for 97.0%. Conversely, two influenza B virus-positive samples were identified among the samples. All other viruses were present at a frequency less than 3%: HRV and ADV in three cases individual (1.1% of 279); HRCV in two cases (0.7%); and HRSV in one case (0.4%). HMPV, FLU-C and HPIV were not detected, even thought the primers were designed to detect these strains. In addition, seven samples (2.5% of the total number of specimens) tested positive for more than one virus or were co-infections: HRSV-A and HRV in two cases; OC43 and FLU-A in one case; HRV and FLU-A in one case; HRSV-A and FLU-A in one case; and a combination of HRSV-A, HRV and FLU-A in two cases. The identity of each virus in these co-infections was confirmed by amplification with primers specific for each virus. Four of the eighteen patients aged 60 years or greater were positive for influenza A virus. For all detected H1N1-positive samples, no viral co-infections were observed.
Among the 279 swabs, 190 (68.1% of 279) samples were subtyped for 2009 influenza A (pH1N1) by the Beijing CDC. The number of respiratory specimens testing positive for influenza by influenza typing is shown in Figure 1. Of the 190 ILI case-patients tested, the positive rates for 2009 influenza A and influenza A(H3) were 5.8% (11/190) and 4.7% (9/190), respectively. Influenza A(H3) was only observed between July and December 2010, and was then replaced by pH1N1. Pandemic influenza A (H1N1) only tested positive in December 2010, and January and February 2011. According to the Chinese National Influenza Center(CNIC), influenza A (H3N2) viruses were the predominant viral etiological factors from June to early January in North China, and from late January, the 2009 influenza A(pH1N1) was the most prevalent, followed by H3N2. Few influenza B viruses (1.7%) were observed during the same period in North China.
Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.
Statistical analyses were performed with Stata® and Excel®. Two seasons were defined to identify possible seasonal trends in circulation of the viruses: winter season during weeks 23 to 39 between June and September and summer season during the rest of the year.
Clinical specimens collected from two sites (HPA Cambridge and WIV-ISP) were screened independently by qualified biomedical scientists for the presence of influenza virus according to their routine testing protocols.
This assay was only used to detect influenza virus types A and B at a higher sensitivity than might be achieved using multiplexed PCR assays, and to further subtype influenza A positive samples. The protocols had been adopted from the studies by Spackman et al.28 and Krafft et al.29 and, respectively, with cycle threshold (Ct) values documented for positive samples (samples with a Ct value less than 40 were considered as positive). Influenza A positive specimens were then subtyped with subtype specific primers and probes that targeted at haemagglutinin (HA) gene of A(H3N2)3, and HA and nucleoprotein (NP) genes of A(H1N1)pdm0930. Cycle threshold (Ct) values were documented for positive samples.
This assay was used to simultaneously detect multiple common respiratory viruses. The SeeGene RV12 kit was used according to the manufacturer’s instructions. The panel included 12 viruses: influenza A, influenza B, respiratory syncytial virus A, respiratory syncytial virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, human metapneumovirus, rhinovirus A/B, adenovirus A/B/C/D/E, human coronaviruses 229E/NL63 and human coronaviruses OC43(referred to henceforth as panel virus).
Clinical specimens were screened for influenza virus with qRT-PCR assays at the regional clinical microbiology laboratory in the Cambridge Health District, United Kingdom. Our previously reported generic quadriplex assay (25, 26) was upgraded for this study to a pentaplex assay capable of detecting all influenza A virus subtypes, influenza B virus, the hemagglutinin A(H1)pdm09 and H3 subtypes, and bacteriophage MS2, and it was performed essentially as outlined in reference 25, with the following modifications. The hemagglutinin H5 subtype primers and probe were replaced with A(H1)pdm09 hemagglutinin-specific primers and probe, H1F, 5′-TCAACAGACACTGTAGACACAGTACT-3′; H1R, 5′-GTTTCCCGTTATGCTTGTCTTCTAG-3′; H1p, Cy5-5′-AATGTAACAGTAACACACTCTGTTAACC-3′-BHQ, with the primer concentrations both at 0.4 μM and the probe at 0.12 μM. An additional set of hemagglutinin primers and probe for H3 seasonal influenza virus was also included, namely, AH3F, 5′-CCTTTTTGTTGAACGCAGCAA-3′, AH3R, 5′-CGGATGAGGCAACTAGTGACCTA-3′, and H3p, VIC-5′-CCTACAGCAACTGTTACC-3′-MGBNFQ, and the primers and probe concentrations were again at 0.4 μM and 0.12 μM, respectively. The generic influenza A virus matrix probe (VIC-5′-TCYTGTCACCTCTGAC-3′-MGBNFQ) was replaced with another generic probe, namely, FAM-5′-CCCCTCAAAGCCGA-3′-MGBNFQ, and used at the same concentration (0.16 μM) along with the AMF primer (0.4 μM) and AMR primer (0.8 μM) as previously reported (25). The reporter label (Cy5) on the influenza B virus probe (BNP probe) was converted to Quasar 705, by accessing the fifth channel (crimson) on the Rotorgene 6000 instrument and creating a pentaplex assay. The concentrations of primers (BNP-F and BNP-R) and the Quasar 705 probe for the influenza virus B component of the assay were, however, increased to 0.2 μM and 0.08 μM, respectively. The MS2 bacteriophage internal control (IC) component was identical to that described previously (25), with the primers (MS2 F1 and MS2 R1) and ROX-labeled MS2 probe concentrations each at 0.08 μM. The pentaplex assay was performed with the use of the SuperScript III Platinum one-step qRT-PCR enzyme (Invitrogen, Paisley, United Kingdom) in a reaction volume of 25 μl (containing 3 mM MgSO4) and the Rotor-Gene 6000 instrument. The amplification conditions were incubation at 50°C for 30 min and at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. Fluorescence was measured for each of the five channels at the end of each cycle. The upgraded pentaplex assay was then subjected to a program of validation to assess its performance before allowing it to supersede our quadriplex assay (25) as our laboratory's front-line diagnostic test. Briefly, 125 routine clinical respiratory specimens, comprising 50 influenza virus A-positive (38 H3N2 and 12 H1N1pdm09), 10 influenza B virus-positive, and 65 negative specimens were processed by both real-time assays in parallel. Concordant results were obtained with both assays for the influenza A and influenza B virus-positive specimens and negative specimens, with the pentaplex assay providing additional typing data for influenza A virus positives, i.e., distinguishing (H1N1)pdm09 from H3N2 isolates. The CT values for the comparable components (generic influenza A and B viruses) of both assays were broadly similar. However, the 12 A(H1N1)pdm09 samples gave a consistently lower CT value, between 1 and 1.5, with the pentaplex assay. The performance of both assays was also assessed using a number of external quality assessment (EQA) panels: the QCMD 2011 influenza hemagglutinin typing EQA program (14 samples) and the QCMD 2011 influenza A and B RNA EQA program (12 samples), obtained from Quality Control for Molecular Diagnostics (www.qcmd.org). The annual HPA Influenza Molecular Proficiency Panel 7 (for 2012), containing 14 samples, was also subjected to both assays in parallel. Both assays delivered a 100% score with the three EQA panels, giving concordant results for the common influenza A and B virus components. The quadriplex assay was, however, able to type the H5 samples, while the pentaplex assay could distinguish and type the H1N1pdm09 and H3N2 samples in the panels, in line with their defining assay attributes. Serial 10-fold dilutions of RNA extracted from virus stocks of A/Solomon Island/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), and B/Panama/45/90 were analyzed by both real-time assays, and their limits of detection were determined to be identical. The analytical sensitivity of the pentaplex assay was calculated using serial dilutions of plasmid constructs containing the matrix gene of A/PR/8/34 (PR8) and the nucleoprotein gene target site of B/Florida/4/2006, and it was found to be ∼5 and 15 genome equivalents per PCR for influenza A and B viruses, respectively.
Double immunodiffusion (DI) for typing of influenza viruses was performed using 1–1.5% agarose. Separate wells were used for typing of influenza A and B in single time using nucleoprotein and matrix protein. Reference antigens and antiserums were diffused in selected wells. Strains that were to be typed were diffused in separate wells for both type A and type B for successful identification of all strains. DID can type virus quite sensitively but virus have to be cultured before typing and low virus titer also limit its use commercially. It is also time consuming and laborious method.
Recommended surveillance strategies included 1) developing an influenza monitoring system and unified national database (both countries) and 2) estimating case-fatality rates (in Argentina only).
Overall, 291 of the 4337 vaccinated individuals returned the questionnaire and reported adverse reactions. This is a rate of 6.7%. The majority of reported adverse reactions was found in the age between 30 and 39 years. (Figure 2)
The most frequently reported local site reactions were: local pain/pruritus or the sensacion of heat at the injection site in 3.8% out of the 4337 vaccinations, myalgia or arthralgia in 3.7%, induration or erythema at the injection site in 2.6%, lymph node swelling in 0.9%, skin rash in 0.3% and ecchymosis at the injection site in 0.1% (Figure 3).
The presence of systemic adverse reactions were reported as follows: fatigue in 3.7%, headache in 3.1%, flu-like symptoms in 2.3%, shivering in 1.9%, temperature > 38°C in 1.3%, dizziness in 1.1%, increased perspiration in 1.1%, gastrointestinal symptoms in 1.0%, drowsiness in 0.9%, , insomnia in 0.7%, formication in 0.3%, Further some severe reportable adverse reactions were observed (0.5%, Figure 3) as one case of facial nerve paralysis, one case of rheumatoid arthritic symptoms and one case of skin alteration which was reported to the local health authorities and the Paul-Ehrlich-Institute.
The clinical and laboratory information of the admitted patients presented as the median and range (minimum to maximum). Statistical significance was assessed using the Student's t-test and the ANOVA test for continuous variables and the χ2 test and the Linear by linear association method for categorical variables. The data were analysed using SPSS version 12.0 for Windows (SPSS Inc., Chicago, IL, USA), and a P value less than 0.05 was considered significant.
ELISA is a very specific and sensitive method for influenza detection. In 1993, a sandwich ELISA was tested in which monoclonal anti-NP antibody attached on plate wells coupled with biotinylated polyclonal anti- A(H1N1)pdm09 whole virus antibodies developed in rabbit was used for detection of nucleoprotein antigen of influenza A virus. This method could detect 10 ng/ml of pure culture. Sensitivity was high because of the use of biotin-avidin signal amplification method. A new sandwich ELISA based diagnostic study was performed during 2009 A(H1N1)pdm09 influenza pandemic. In this ELISA, specific monoclonal antibodies and horseradish-peroxidase linked rabbit anti-HA polyclonal antibodies against HA protein were used. This kit was developed by Xiamen University, China. They reported overall sensitivity (0.57) was higher of ELISA than rapid influenza diagnostic kit QuickVue Influenza A+B test (0.43). The sensitivity of both test varied according to the different viral load. It was 100% at high viral load and then decreased with low viral load. ELISA sensitivity decreases after 105 (log 10/ml) viral load but QuickVue Influenza A+B test sensitivity decreases after 107 (log 10/ml) viral load. In a different study, 1086 sera were analysed from 43 swine herds for different reference strains (H1N1, H3N2, H1N2, H1N1pdm) using ELISA with HI test and it was found that ELISA sensitivity and specificity were higher than HI that were 72.65% and 63.01%, respectively, for the detection of these strains. According to these studies, ELISA cannot detect infection at an early stage. A new ELISA-based immunosensor was developed using AuNP (gold nanoparticles) for improved sensitivity. EDC-NHS was used for the binding of anti-HA with AuNP that was already treated with a layer of formic acid (HCOOH). A(H1N1)pdm09 viruses having HA on surface binds with antibodies and secondary antibodies conjugated with (+) AuNP against NA antigen were used. Electrostatic interactions between nanoparticles and antibodies help in increasing the surface area for antibodies that helps in increasing sensitivity. More the number of antibodies binds with nanoparticles, and more antigens can be captured that help in increasing the sensitivity of the procedure. The catalytic activity of gold nanoparticles toward TMB-H2O2 makes it oxidized, which further causes a change in colour and gives a signal.
The mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days.
A multiplex PCR panel able to identify FLU A and B, Human Metapneumovirus, Adenovirus, Coronavirus 229E/NL63, Parainfluenza viruses 1, 2, and 3, Coronavirus OC43, Rhinovirus A/B, and Respiratory syncytial A and B (RV12 ACE Detection 23 Seegene, Seoul, Korea), using the dual priming oligonucleotide (DPO) system, was used according to the manufacturer’s instructions. An internal control included in the primer mixtures was used to assess any potential PCR inhibitory effects. All of the samples that tested positive for FLUA or FLUB were further characterized by genotyping analysis to identify the subtype. For FLUA subtyping was performed using a procedure used in our laboratory, instead for FLUB a homebrew procedure was set-up according to WHO recommendations.
The present study was performed to compare a custom multiplex assay and an FTD multiplex assay by testing of 356 respiratory samples obtained from children with SARI admitted in J K lone paediatric hospital Jaipur.
In the present study, the concordance between the custom assay and the FTD assay was found to be 100 % for Flu A, Influenza A(H1N1) pdm09, HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, and HPeV. Similarly Chen et al., reported a concordance of 99.60 % for Flu A and Influenza A(H1N1) pdm09 when comparing a multiplex PCR assay with a uniplex assay.
The concordance between the two assays varied from 94.66 to 99.71 % for the remaining ten viruses; Flu B (99.71 %), HPIV-3 (99.71 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), HCoV HKU1 (99.71 %), HAdV (99.71 %), HRV (99.71 %), EV (98.31 %). Similar findings have been observed in earlier studies for Flu B (98.25 to 99.42 %), HPIV-3 (96.53 to 99.30 %), HPIV-4 (97.10 %), HCoV NL63 (95.95 to 100.0 %), HMPV A/B (99.65 to 100.0 %), RSV A/B (93.06 to 98.60 %), HCoV HKU1 (98.84 to 100.0 %), HAdV (97.20 to 100.0 %) [8, 9]. Concordance for EV in the present study was different from an earlier study (93.00 %). The difference in concordance obtained in different studies may be due to the different primer binding regions or may be due to different methodologies employed by various studies. The number of samples positive for HCoV 229E, HPIV-4, HPIV-2, HCoV NL63, HPeV, HCoV HKU1, Flu A, were ≤ 5 in the present study. Studies based on larger numbers of samples are required to assess the concordance of these viruses more thoroughly.
The limit of detection for some of the viruses in the custom assay (Table 7) ranged from 1 DNA copy/ml to 2×104 copies/ml [7, 10–14]. The detection limit of the FTD assay for different viruses was 102 copies/ml for FluA, HPIV-2, HMPV and HCoV OC43; 103 copies/ml for FluB, HCoV HKU1, HPIV-1, HBoV, HPIV-3, HCoV NL63, RSV, HAdV, EV, and HPeV; and 104 copies/ml for HRV, HCoV 229E and HPIV-4.
In the present study RSV A/B was the most predominant virus detected by both the custom and FTD assays with positivity in 84 (23.60 %) and 67 (18.82 %) samples respectively and concordance of 94.66 %. This finding is different when compared with other studies [8, 16] where comparisons were made between multiplex PCRs in which RSV was the second most predominant virus detected.
The custom primer and probes used for Influenza A(H1N1) pdm09, RSV A/B, Flu B, HMPV A/B, HBoV, HRV, HPIV-1-4, HAdV and HCoVs showed a positivity of 7.58, 23.60, 3.65, 11.80, 4.49, 18.54, 11.79, 7.58 and 3.93 % respectively for each virus in the present study in comparison to a positivity of 18.39 %, 14.1 %, 13.3 %, 2.9 %, 0.5–4.5 % [13, 18, 19], 20.78 %, 8.62 %, 3.5 %, and 4.70 % respectively in earlier studies where the same primer and probes were used. HBoV was mostly associated with co-infections in the present study in both assays. This is consistent with an earlier study
The major discrepancy in the present study was found with RSV A/B. The discrepancy in 18 samples which were over detected by the custom assay was resolved by RSV A and RSV B typing. The RSV typing results for the discrepant samples showed that all 18 samples were RSV B. Further all samples positive for RSV A/B by the FTD assay were also subjected to RSV typing which indicated RSV A in 13 (19.40 %) samples, RSV B in 50 (74.63 %) samples and RSV A & RSV B dual infections in 4 (5.97 %) samples
During the process of standardisation of the custom assay 3 μl of viral nucleic acid (positive control) was used for each virus including 4 picomoles of primers and 2 picomoles of probes. Each panel consisted of 3 viruses. In total 9 μl of viral nucleic acid was used for each panel. While the FTD assay used 10ul of nucleic acid in each tube with primers and probes for 4 viruses, the concentration of primer and probe was not disclosed by FTD. In total 4 μl more of viral nucleic acid was used in the custom assay compared to the FTD assay which may have increased the sensitivity/detection of different viruses in the custom assay.
Initially during the process of standardisation of the custom assay, different primer and probe concentrations were tried and the PCR was run for 45 cycles as per the protocol followed by various authors. Although data was analysed using PCRs run for 35 and 40 cycles, best results were achieved using a Ct value of 35 for both the FTD assay and the custom assay. Accordingly, a Ct value of <35 was considered as positive for both assays as per the FTD kit. With the custom assay being run for 40 cycles this reduces the custom assay run time by 8 min, thereby making it only 21 min longer than the FTD assay.
Comparisons were made between various aspects of the custom and the FTD assays (Table 6). No major differences were observed between the two assays except in the cost incurred for both assays. Similar comparisons were also done in an earlier study where three multiplex PCRs were compared. The turn-around time of the custom assay was 29 min more as compared to the FTD assay. But both the assays reported the results on the same day. The excess time of 29 min taken by the custom assay as compared to the FTD assay may not greatly interfere with the treatment process. However, the custom assay was much more economical costing INR 1500/- per sample for screening 18 respiratory viruses compared to the commercial FTD assay which was expensive costing INR 4300/- per sample. This assay may prove to be highly cost effective in resource limited settings like ours. However the limitation of our study was that some of the viruses showed low positivity as a result it is difficult to assess the concordance accurately. Larger numbers of positive samples need to be tested to evaluate the concordance of these less prevalent viruses.
The burden of influenza in children is substantial and hospitalization rates are highest among the youngest children (16, 17). Notably, half of flu-related hospitalizations and deaths occur in previously healthy children, so a vaccination strategy that only includes children with comorbidities does not seem to be effective (18).
Influenza immunization in children is the most effective way to prevent disease, affording both direct and indirect protection. The protective effect of the vaccine depends largely on the match between vaccine strains and circulating viruses. As children are usually the family members who bring influenza into their household, vaccinating children could help mitigate outbreaks (19). One health care model showed that vaccinating 20% of schoolchildren had a greater impact on flu-related mortality than vaccinating 90% of the elderly (20). When influenza immunization of schoolchildren was introduced in Japan, all-cause deaths, as well as influenza and pneumonia-related deaths, were significantly reduced in all age groups (21), and when vaccination was stopped, mortality rates increased, especially in the elderly.
There are, however, several hurdles to introducing an early childhood influenza control program. First, there are limited vaccine options. IIV3s have moderate efficacy in young children (22), and do not induce persistent immune response. Inactivated influenza vaccines, quadrivalent (IIV4s) offer broader protection against the two B lineages, which usually co-circulate yet alternate in dominance (2). Although B viruses predominate in children, they are a significant cause of hospitalizations and deaths in all age groups. Use of IIV4 could reduce a mismatch between a vaccine and circulating B lineages, but the benefits would be modest (23). Adjuvanted IIV3s (aIIV3s) are more effective than non-adjuvanted vaccines. In a trial of MF59-adjuvanted IIV3 in 4 700 children 6–72 months old (24), efficacy was 85% (the highest ever reported in children 6–24 months old) and persisted after the second dose, and there were no safety issues. According to one study, the correlate of protection threshold is higher in children than in adults (25); for example, titers of 1:110 in children and 1:40 in adults were both associated with 50% clinical protection. In Canada, aIIV3 is licensed for use in infants and young children 6–24 months old due to its superior immunogenicity and acceptable safety profile (26). At the roundtable meeting, use of the live attenuated vaccine was also discussed, but as this vaccine is not available in Argentina or Brazil, the information was not included in this report.
A second hurdle to introducing a childhood influenza control program is related to safety and reduced confidence due to adverse events following immunization (AEFIs), a problem mostly seen in Europe and North America. Unexpected AEFIs with two flu vaccines have had a negative impact (27, 28). In addition, poor vaccine performance could discredit the integrity of the NIP and dampen the success of other routine vaccines.
A third hurdle concerns funding requirements. Introducing flu vaccine into an NIP requires evidence of cost-effectiveness involving high vaccine efficacy; reasonable cost (with drawbacks including the need for two-dose priming and/or annual revaccination); and negligible AEFI costs. Currently, 29 countries in Latin American but only seven in Europe have routine influenza childhood immunization programs, and vaccine uptake in developed countries does not surpass 30%.
At the meeting, various solutions were suggested to overcome these hurdles, including 1) using more efficacious vaccines; 2) building greater confidence in safety by extending post-licensure surveillance; and 3) making public programs more flexible by broadening the interval of the two-dose schedule, irrespective of the season, and expanding school delivery programs to include children 1–5 years old.