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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.
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.
The majority of elderly individuals (≥60 or ≥65 years old) and those with serious co-morbidities would be candidates for influenza vaccination even without travel plans. However, it remains important to assess vaccination status and to evaluate whether or not the strains the individual has been vaccinated against are appropriate for the geographic area and season of the travel plans (see below). Recommendations for the elderly are not only based on the inevitably growing number of co-morbidities in this age group, but also on immune-senescence.62–64 Unfortunately, the effectiveness of influenza vaccines is often impaired in individuals who could benefit most from vaccination: immune-compromised and elderly individuals, as well as patients in the other high-risk groups mentioned in virtually all recommendations. Limited data are available about the added value of recently introduced adjuvanted, high-dose (HD) and quadrivalent vaccines (for review see Reperant et al.65). To date, strong RCT data providing evidence of superiority are only available for the HD formulation in the elderly and the effect was modest.66
Although in the past one has been reluctant to immunize pregnant women, currently vaccination of pregnant women against seasonal influenza is incorporated in most guidelines and recommendations. This is based on the real risks of influenza during pregnancy that by far outweighs the risks associated with vaccination. During the H1N1 (2009) pandemic, influenza vaccines proved to be safe and effective for pregnant women and their unborn babies; findings very similar to studies of seasonal influenza vaccination in this high risk group.67,68 In line with these recommendations, the advice might be broadened to pregnant women travelling to influenza endemic areas and possibly to persons in close contact with pregnant women or other high-risk individuals, like partners and close family members [CDC guideline: http://www.cdc.gov/h1n1flu/clinician_pregnant.htm (26 October 2016, date last accessed)] or travel partners.
Influenza vaccination status of travellers in defined risk categories should be checked and either vaccination or additional vaccination against influenza should be recommended on the basis of the epidemiological situation in the area of intended travel. The relatively limited effectiveness of influenza vaccination in most of the high-risk groups and the value of newer generations of vaccines that might overcome these problems are important topics for future study. Finally, for very frail patients, the advice not to travel to certain areas should always be considered during pre-travel consultation, although risk assessment in these cases should obviously not be limited to the threat posed by influenza.
Currently, seasonal influenza vaccination in most guidelines is only advised for healthy travellers if they plan to attend large events or to travel by cruise ship. This is mainly because influenza is widely considered a relatively mild and self-limiting disease in most healthy individuals.2 However, over the past decade, reports of patients without co-morbidities who develop severe and even lethal influenza with apparently ‘normal’ seasonal influenza viruses, have steadily accumulated.7,32,36,37,59,60. Since seasonal influenza is the most frequent vaccine preventable infectious disease in travellers, influenza vaccination should be part of regular pre-travel advice for all travellers. This raises the more general question about what burden of expected disease during the envisaged travel would justify inclusion of vaccination advice in travel guidelines. The probability of acquiring influenza, severity of disease, expected effectiveness of the vaccine and cost are among the factors that should be taken into account. One could argue that the a priori chances of developing typhoid fever, hepatitis A or tetanus during a two and half week trip to an Asian destination are much lower than being infected with an influenza virus. However, at least according to most guidelines issued in industrialized nations, these three immunizations are usually recommended for most travellers going to many developing countries while influenza vaccination is often not even considered.61
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.
Anti-viral drugs are thought to be backbone of a management plan of an avian flu pandemic. Only two anti-viral drugs have shown promise in treating avian influenza: oseltamivir (Tamiflu®) and zanamivir (Relenza®). A treatment of Tamiflu® includes 10 pills taken over five days while Relenza® is administered by oral inhalation. The US Food and Drug Administration has approved both anti-viral drugs for treating influenza but only Tamiflu® has been approved to prevent influenza infection. Because antivirals can be stored without refrigeration and for longer periods than vaccines, developing a stockpile of antivirals has advantages as part of an overall strategy to control a flu epidemic. However, there are limitations to the use of antivirals: Tamiflu® needs to be taken within 2 days of initial flu symptoms for it to be effective, but many people may not be aware that they have the flu early in the disease. Some research in animals and recent experience in the use of the drug to treat human cases have also found that Tamiflu may be less effective against the recent strains for the current H5N1 virus than the 1997 strain. Improper compliance to antivirals by irresponsible individuals during an outbreak may results in the emergence of a drug-resistant strain. Lastly, there are current concerns about the safety of Tamiflu® which has been associated with increased psychiatric symptoms among Japanese adolescents.
A multitude of scientific and technological advances have occurred over the past century, allowing for a greater understanding of the dynamic relationship between the host and influenza viruses during infection. These advances, along with access to autopsy samples and the reconstitution of the 1918 pandemic virus, have facilitated a greater understanding of how the pandemic virus differs from other seasonal and pandemic influenza virus strains. Moreover, technological advancements following the 1918–1919 influenza pandemic virus have facilitated the development of preventative measures, including vaccines and antivirals, to limit widespread illness due to influenza infections.
The determination of the genomic sequence of the 1918 pandemic virus, and the subsequent reconstruction of the virus, has provided us with the opportunity to decipher the viral- and host-specific properties that contributed to the severity of the 1918–1919 pandemic. It has been demonstrated that in contrast to other influenza viruses, the 1918 pandemic virus is highly virulent and pathogenic in multiple animal species without prior adaptation [45, 50]. While obvious knowledge gaps remain, in particular with respect to the origin of the virus and the molecular mechanisms (host and/or viral) underlying differential pathogenesis as compared to other influenza viruses, there have been considerable advances in our understanding of the 1918 pandemic virus.
Since the isolation of the first human influenza virus in 1933, researchers have worked to develop an effective influenza vaccine. Current influenza vaccines are reformulated seasonally and provide protection against circulating influenza A and B viruses. The World Health Organization conducts worldwide surveillance studies throughout the year on currently circulating influenza strains, and thus recommends which strains should be included in each influenza vaccine. While the seasonal influenza vaccine is approximately 60% effective, this protection is dependent on the characteristics of the individual being vaccinated, including age and overall health, as well as the match between the strains included in the vaccine formulation and currently circulating strains. Individuals who have been vaccinated are generally protected from illness and provide a measure of protection for those who are not able to be vaccinated due to their age or other health issues through herd immunity. There has also been increasing interest in the development of “universal” influenza vaccines designed to provide protection against a wide range of antigenically-distinct influenza viruses, including those currently in circulation and those that may emerge in the future. These will not be discussed in detail as recent reviews have provided excellent discussions of this topic [51–57].
Two major classes of antivirals have emerged for therapeutic treatment of severe influenza virus infections. Adamantane antivirals target the matrix-2 (M2) surface protein, while neuraminidase (NA) inhibitors target the NA viral surface protein. Adamantane compounds were the first licensed influenza antivirals and block the M2 ion channel protein from properly functioning, thus effectively blocking membrane fusion [58, 59]. Unfortunately, adamantane antivirals are only able to target influenza A viruses limiting their application for influenza B virus infections. Further, more than 90% of influenza A viruses are resistant to this class of drugs due to the high mutation rate of the virus [58, 60]. Thus, the use of NA inhibitors is recommended. NA inhibitors block the NA surface protein and prevent the release of progeny virus and infection of additional cells. While resistance to NA inhibitors has been observed in some influenza virus strains, they are still highly effective in the majority of patients. Studies have shown that both adamantane antivirals and NA inhibitors provide protection against the 1918 virus.
Although outside the auspice of this commentary, it should be mentioned that advances in mechanical ventilation modalities, including non-invasive positive pressure ventilation, from the 1950s onwards, have provided an additional support mechanism for treatment of severely ill patients. The routine clinical use of antibiotics in the early twentieth century also heralded a new era for combating influenza viruses. As a testament to this, excess influenza mortality declined significantly from 1942 to 1951 onwards [61–63]. However, the widespread general administration of antibiotics has resulted in an escalating public health crisis due to multi-drug resistance. This has impacted the treatment of severe influenza infections, as methicillin-resistant S. aureus (MRSA) is the most frequently isolated bacteria from patients with severe influenza-bacterial co-infections in the US [64, 65] and complicated up to 55% of fatalities during the 2009 pandemic [66–69].
A total of 73 influenza-positive (confirmed using a qRT-PCR method) clinical nasopharyngeal aspirate samples collected from patients who demonstrated flu-like symptoms at Chungbuk National University Hospital, Republic of Korea were used for clinical evaluation of the RT-LAMP diagnostic assay developed in this study. In addition, 18 spiked samples in which 104 TCID50/ml of viruses (i.e., H1N1, H5N6, H5N8, and H7N9) were diluted into flu-negative human nasopharyngeal aspirate samples were used for clinical evaluation. RNA was extracted from clinical/spiked samples and subjected to multiplex RT-LAMP and conventional RT-PCR for comparative detection of specific influenza viruses. To verify the specificity of the RT-LAMP assay using clinical samples, 44 RNA samples of human infectious viruses other than those targeting influenza viruses including human enterovirus (HEV), adenovirus (AdV), parainfluenza virus (PIV), human metapneumovirus (MPV), human bocavirus (HboV), human rhinovirus (HRV), human coronavirus 229E (229E), human coronavirus NL63 (NL63), human coronavirus OC43 (OC43), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB), and Middle East Respiratory Syndrome coronavirus (MERS-CoV) were subjected to multiplex RT-LAMP assay. RNA samples of all other human infectious viruses except MERS-CoV were extracted from clinical swab or nasopharyngeal aspirate samples collected from patients and confirmed positive by qRT-PCR. RNA samples from MERS-CoV grown in cell culture were extracted and diluted in human nasopharyngeal aspirate as additional samples for spiked test.
To determine the specificity of the optimized multiplex RT-LAMP assay for targeting tested influenza viruses (type B, H1, H3, H5, and H7), RNA samples of other subtypes of influenza viruses including A/Em/Korea/W357/2008 (H2N3), A/Em/Korea/W210/2007 (H4N4), A/Em/Korea/W502/2015 (H6N2), A/Em/Korea/W563/2016 (H8N6), A/Em/Korea/W233/2007 (H9N2), A/Em/Korea/W372/2008 (H10N7), A/Em/Korea/W552/2016 (H11N9), and A/Em/Korea/W373/2008 (H12N5) were assessed using the multiplex RT-LAMP assay described in this study. In addition, one-step RT-PCR or qRT-PCR was performed to confirm the presence of viral genomic RNA using specific primer sets (Additional file 1: Table S1). One-step RT-PCR conditions were the same as described above. Results of one-step RT-PCR were confirmed using 2% agarose gel electrophoresis.
A structural questionnaire was designed to investigate: (1) AI awareness, (2) knowledge of government policies, and (3) protection measures used. To achieve the study objective, the team members who designed and reviewed the questionnaire included infectious disease physicians, infectious disease epidemiologists, scholars experienced in knowledge, attitude and practice (KAP) of diseases, field workers who frequently went to LPM to take poultry specimens, and administrators in LPMs. The questionnaire included items such as demographic information, job duties, prevention measures, personal perceptions, the impact of China’s AI outbreaks on Taiwan, attitudes toward different policies such as killing poultry at LPMs, and potential confounding variables (age, gender, educational level, and living area) [Additional file 1: Appendix 1, Additional file 3: Appendix 3]. We did a pilot test on both study groups in different geographical areas to assure full understanding and reliability. After a comprehensive review by questionnaire design team members, the wording of the questionnaire was revised and simplified to maximize the response rates. There were five main questions measuring risk awareness, attitudes and personal protection measures (RAP) [Additional file 3: Appendix 3]. In addition to these five main questions, questions on the awareness of HPAI H5N2 outbreaks in 2012 and risk perception in LPAI-H5N2, HPAI-H5N2 and other important infectious diseases in Taiwan [such as severe acute respiratory syndrome (SARS)] were also included in the questionnaire of the second surveys for better comparison. Most questions were multiple choice, with a comprehensive range of choices or differential scales or rankings [Additional file 4: Appendix 4]. However, the second main question on possible future outbreaks of human cases of infection with AIVs in Taiwan was measured by the Likert scale. The questionnaire was administered by well-trained interviewers.
Using viral RNA of A/equine/Kildare/2/2010, the detection limits of the molecular tests, that is conventional RT‐PCR, rRT‐PCR and RT‐LAMP, for EIV were 102·8EID50/ml, 102·1EID50/ml and 102·8EID50/ml, respectively.
The comparison of detection limits of the 22 RAD tests for EIV is shown in Table 1. The detection limits of the four tests, ImmunoAce Flu, BD Flu examan, Quick chaser Flu A, B and ESPLINE Influenza A&B‐N, were 104·9EID50/ml. The detection limits of Prime check Flu・RSV, Clearview Exact Influenza A&B and POCTEM S influenza were 106·3EID50/ml. The detection limit of Gold sign FLU was 107·0EID50/ml. Clearline Influenza A/B/(H1N1) 2009 and QUICKVUE Rapid SP influ did not detect EIV in the undiluted stock virus. The detection limit of the other twelve tests was 105·6EID50/ml.
The four most sensitive RAD tests and Prorast Flu as an example of a less‐sensitive RAD were evaluated in an EI experimental infection study, and their diagnostic sensitivity was compared to those of VI and molecular diagnostic assays. The results obtained by the different assays for the sequential samples collected from the horses experimentally infected with A/equine/Kildare/2/2010 are summarized in Table 2.
The average detection periods for the assays are illustrated in Figure 1. The mean duration (days) of positive rRT‐PCR results was significantly longer than those of all the RAD tests (P < 0·001 to =0·034). The mean duration of RT‐LAMP‐positive results was significantly longer than those of the RAD tests (P < 0·001 to =0·011), except for ImmunoAce Flu (P = 0·05) and Quick chaser Flu A, B (P = 0·073). The mean duration of conventional RT‐PCR‐positive results was significantly longer than those of the RAD tests (P < 0·001 to =0·025), except for ImmunoAce Flu (P = 0·087) and Quick chaser Flu A, B (P = 0·143). The mean duration of positive Prorast Flu results was significantly shorter than those of all the other tests in this study (P < 0·001 or 0·011). There was no significant difference in the mean durations of positive results obtained with the molecular assays (P = 0·502–0·575).
The evaluation of the performances of the most sensitive RAD tests to diagnose EI in field samples is summarized in Table 3. All rRT‐PCR‐negative nasopharyngeal samples were negative by the RAD tests. Of the 30 rRT‐PCR‐positive samples, BD Flu examan detected 22 (73%) and Quick chaser Flu A, B and ImmunoAce Flu detected 20 (67%) as positive. ESPLINE Influenza A&B‐N was less sensitive and detected only eight positive samples (27%). Kappa coefficient values indicated a substantial agreement between BD Flu examan, Quick chaser Flu A, B and ImmunoAce Flu and rRT‐PCR but only a fair agreement between ESPLINE Influenza A&B‐N and rRT‐PCR.
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.
The average detection periods were analysed by repeated‐measures analysis of variance and post hoc Student–Newman–Keuls method, using sigmaplot 11.2 (Systat Software, San Jose, CA, USA). A level of P < 0·05 was considered significant. Analysis of performance of the RAD tests with the clinical samples, sensitivity, specificity, kappa coefficient values were calculated using Excel 2010 (Microsoft Japan, Tokyo, Japan). The kappa coefficient values were evaluated according to the guideline mentioned by Landis and Koch25 as follows: <0 as no agreement and 0–0·20 as slight, 0·21–0·40 as fair, 0·41–0·60 as moderate, 0·61–0·80 as substantial and 0·81–1·0 as almost perfect agreement.
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.
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.
The RPM-Flu assay panel simultaneously addresses 30 different categories of viruses and bacteria that are more or less likely to be encountered in specimens related to respiratory infections (Figure 1, Figure S1). Each assay simultaneously enables detection and identification of targeted pathogens from one or more of 188 different target gene resequencing detector tiles.
For comparison, the recently FDA-cleared Luminex® Respiratory Viral Panel (RVP) Multiplex Nucleic Acid Detection Assay Respiratory Virus Panel (510(k) K063765) is a twelve-plex RT-PCR panel for detection and differentiation of a few strains and types of respiratory viruses including adenovirus, influenza A virus (as A/H1 or A/H3), respiratory syncytial virus, parainfluenza virus, metapneumovirus and rhinovirus (no bacterial respiratory pathogens tested). The RVP assay yields no specimen-specific viral gene sequences that could be used for more detailed identification and differentiation of detected virus strains and variants of detected virus(es).
For human and avian type A influenza viruses in particular, the RPM-Flu provides direct detection and identification capability for any of the combinatorial 144 A/HN subtypes that may be present in a specimen, using an ensemble of 39 different influenza virus target gene resequencing detectors. The Luminex RVP multiplex RT-PCR panel uses three of its twelve primer-probe sets to detect type A influenza virus (M gene-specific primer pair) and distinguish A/H1 from A/H3 (HA1- and HA3-specific primer pairs). While this RT-PCR panel may infer influenza virus subtype, not even testing for the NA component, the assay result is only based on a pattern of signals from a small panel of RT-PCR primer-probes.
Some RT-PCR protocols require serial analysis with hierarchical panels of primer-probes in order to expand the practical multiplicity of pathogen targets or variants. This is the case with the benchmark PCR testing used in this report –a type A influenza virus-positive specimen may be identified in a first round RT-PCR panel, followed by testing with another panel to distinguish A/H1 from A/H3. Failure to establish subtype A/H1 or A/H3 in the follow-on test is a first tier screening results for possible identification of the 2009 Novel A/H1N1 influenza virus outbreak strain. According to the FACT SHEET FOR HEALTHCARE PROVIDERS: INTERPRETING CDC HUMAN INFLUENZA VIRUS REAL-TIME RT-PCR DETECTION AND CHARACTERIZATION PANEL FOR RESPIRATORY SPECIMENS (NPS, NS, TS, NPS/TS, NA1) AND VIRAL CULTURE TEST RESULTS (Authorized by FDA 02 May 2009),
It is not at all clear that such hierarchical RT-PCR protocols, attempting to increase analytical multiplicity through serial testing of the same specimen(s) at different laboratory venues over a period likely longer than a single day can be either efficient or cost-effective. Far more diagnostic intelligence about the patient and specimen would be available in first-day same-day results from a single RPM-Flu test of the original specimen.
Currently, the H5N1 avian flu virus is limited to outbreaks among poultry and persons in direct contact to infected poultry. Avian influenza (AI) is endemic in Asia where birds often live in close proximity to humans. This increases the chance of genetic re-assortment between avian and human influenza viruses which may produce a mutant strain that is easily transmitted between humans, resulting in a pandemic. Unlike SARS, a person with influenza infection is contagious before the onset of case-defining symptoms. Researchers have shown that carefully orchestrated of public health measures could potentially limit the spread of an AI pandemic if implemented soon after the first cases appear. Both national and international strategies are needed: National strategies include source surveillance and control, adequate anti-viral agents and vaccines, and healthcare system readiness; international strategies include early integrated response, curbing disease outbreak at source, utilization of global resources, continuing research and open communication.
Continuous variables were summarized as mean (±SD) or median (with interquartile ranges). For categorical variables, the percentages of patients in each category were calculated. Unpaired t test was used in normal distribution parameters comparing the mean values of the parameters of the two groups. A p value of less than 0.05 was considered to indicate statistical significance. Characteristics of the groups and clinical laboratory parameters were compared between the two groups (A, B). All analyses were carried out with the use of SPSS statistical software package (SPSS version 17.01; SPSS, Chicago, IL, USA).
All specimens described in this report that were originally obtained from human subjects were obtained with informed (verbal) consent of study participants allowing for further research use, following Institutional Review Board-approved research protocol NHRC.1999.0002, Triservice Population-Based Surveillance for Respiratory Pathogens Among High-Risk Military Personnel, CAPT Kevin Russell, MC, Principal Investigator. The NHRC IRB approved verbal consent for this protocol because of minimal risk to volunteers. Samples are collected for accredited diagnostic testing, and only used for further research in a de-identified manner with minimal demographic data (date and site of collection, age, sex), tested only for respiratory pathogens and analyzed in aggregate, and resulting data is not used for patient treatment or management. No part of this study was conducted as experimentation involving live vertebrate animals. Reference strains of avian influenza viruses were propagated in laboratory cultures for analysis of viral RNA.
Multiplex PCR‐based NAATs have been increasingly used for syndromic diagnosis, due to their high throughput, high sensitivity, high specificity, cost‐effectiveness, and great clinical significance.10, 11, 12 The ResP assay is based on multiplex PCR amplification and capillary electrophoretic separation of PCR amplicons by length. This technique has been used for pathogen detection and subtype classification of pediatric acute lymphoblastic leukemia.13, 14 By comparing the results with a standard size marker of targeted pathogens, pathogens in samples can be separated and identified as expected.15 The subtypes of most viruses were not designed to be further distinguished by this assay, except for influenza virus A. The influenza virus A pdmH1N1 (2009) and H3N2 are the two subtypes which are most popular in China recently. Therefore, a patient whose specimen is positive for influenza virus A but negative for influenza virus A pdmH1N1 (2009) or H3N2 is probably infected by an uncommon influenza virus A, such as H7N9, H5N1, H5N6 avian influenza virus A16, 17, 18 and has to be immediately quarantined once it is confirmed. It should be noted that hospitals, not CDCs, are the first to reach such patients, so this assay helps hospitals identifying such high‐risk patients and make appropriate quarantine measurement in a timely manner to control further spread of avian influenza A virus.
This assay has previously been clinically applied to detection of respiratory pathogens in hospitalized children suffered with community‐acquired pneumonia (CAP)14 or lower respiratory tract infections.19 The assay was evaluated by comparing with Sanger sequencing, showing great performance with 100% positive prediction value (PPV) and 99.85% negative prediction value (NPV).20 To our knowledge, this is the first study evaluating the performance of the ResP in oropharyngeal swab specimens from outpatients with ARIs.
Our study showed almost perfect kappa statistics for the ResP on rhinovirus, adenovirus, influenza virus A pdmH1N1(2009), respiratory syncytial virus, and influenza virus B, suggesting that the performance of ResP on these viruses was as effective as pathogen‐specific PCRs. On human metapneumovirus, the kappa statistics were lower than 0.8, presumably due to the small number of positive cases. Overall, this assay demonstrated 86.5% PPV and 97.8% NPV. This work suggested that the performance of ResP was sufficient enough be used for respiratory pathogen identification in outpatients with flu‐like manifestations.
The major limitation of this study is the small number of human metapneumovirus, parainfluenza virus, Mycoplasma pneumoniae, boca virus, influenza virus A H3N2, coronavirus, and Chlamydia. Further investigation is needed to evaluate the performance of ResP on these pathogens.
In conclusion, the performance of ResP showed a high‐degree agreement with pathogen‐specific PCRs in oropharyngeal swabs from outpatients. The implementation of ResP may facilitate the diagnosis of respiratory infections in a variety of clinical scenarios.
With the aim of improving prevention and control of viral outbreaks, the Chinese government has been investing continually in the advancement of science and technology since 2003, including the appropriation of more than 12 billion RMB for research and development related to combating SARS, influenza, and other major infectious diseases. Meanwhile, China has built 11 national technology platforms, 11 national research centers, 6 national key laboratories, and 2 national engineering laboratories. In 2010, the Chinese National Influenza Centre was designated as a WHO Collaborating Centre for Reference and Research on Influenza. All these laboratories and funding contributed to application of advanced technologies in preventing and controlling infectious diseases.
Above all, quick identification of pathogens is a prerequisite to controlling emerging epidemics. To achieve it, China has developed state-of-the-art pathogen isolation and identification technologies such as high-throughput sequencing method. In contrast to the SARS-Cov debacle, H7N9, H10N8, and H5N6 were identified within China [28–30]. BGI, a Chinese company, helped Germany sequence the pathogen Escherichia coli O104:H4 within a week using high-throughput sequencing technology in 2011. Meanwhile, Chinese researchers exploring the genesis and source of emerging viruses have found that bats are natural reservoirs of SARS-like coronaviruses and have demonstrated that domestic fowl play an important vector role for H5N1 and H7N9 [4, 32, 33].
The government encourages the development of diagnostic reagents, vaccines, and medicines as well as prophylactic equipment (e.g., infrared thermometers). China's national vaccine regulatory system was confirmed to meet WHO standards in 2011. China has developed SARS, H5N1, H1N1, and H7N9 vaccines (Table 1) and became the first country to use an H1N1 vaccine. China now produces oseltamivir (like Tamiflu®) and peramivir (like Rapivab®), obviating the need to import antivirals.
China's improvements in research funding and technical capabilities have led to a series of important findings. For example, Chinese researchers have revealed the crystal structures of key viral proteins (e.g., SARS-Cov protease, H1N1 neuraminidase N1, and H5N1 polymerase PAC-PB1N complex) [36–38], which is useful for drug design, and discovered an oseltamivir-resistance mechanism in H7N9. A traditional Chinese medicine (TCM) herbal formula was confirmed to reduce H1N1 influenza-associated fever safely and with efficacy similar to that of oseltamivir in a randomized clinical trial.
A total of 420 oropharyngeal swabs were enrolled from 10 hospitals and 10 CDCs in Guangzhou from 2017 to 2018. Samples were collected from a wide range of ages, with the average age of 27.2 (Table 1). About 55% specimens were from male.
A pathogen‐positive result was determined when the pathogen‐specific fragment(s) was positive, as shown in Figure 1. A negative result was determined when none of the 13 pathogen‐specific fragment was positive, while the controls (huDNA, huRNA, and IC) were positive (Figure 2). In this study, the ResP detected positive results in 141 samples, accounting for 33.6%, while the comparator tests detected positive results in 127 samples, with positive rate 30.2%. Among the detected pathogens, rhinovirus was the most common, followed by adenovirus and influenza virus A pdmH1N1 (2009) (Table 2). Of the 420 specimens, the ResP yielded consistent positive results in 121 specimens (86.5%, 121/141), and consistent negative results in 273 specimens (97.8%, 273/279) comparing with pathogen‐specific PCRs, leading to an overall agreement of 93.8%.
No specimen was detected positive with coronavirus or Chlamydia. In six of the ten detected pathogens, the Cohen's kappa values were over 0.8 with P value <.01 (Table 2). The lowest kappa (0.70) was observed on human metapneumovirus.
The WHO Vaccine Composition Meeting for the 2014-2015 season held in February 2014 in Geneva approved that A/California/7/2009 (H1N1)pdm09-like virus, A/Texas/50/2012 (H3N2)-like virus, and B/Massachusetts/2/2012-like virus should be included in the trivalent vaccine formulation in the Northern Hemisphere.52 Consequently, the aforementioned novel avian strains are out of scope of the current vaccine formulation indicating the severity of the situation if the H7N9 influenza has global dissemination.
Laboratory testing in preliminary cases showed that neuraminidase inhibitors (oseltamivir, zanamivir) were effective against H7N9 infections, but the adamantanes were not. In addition, early treatment with neuraminidase inhibitors have been reported to restrict the severity of illness.34,53