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HA, the major surface protein of IAV, mediates the viral binding, membrane fusion and viral entry. Determination of the crystal structures of HA0 provides important information for understanding the pH-induced conformational changes of HA at the pre-fusion, intermediate and post-fusion states for development of anti-influenza drugs. Although a series of anti-influenza drugs targeting the NA and M2 ion channel are currently available, the emergency of drug-resistance viruses has raised the great concern on their ineffectiveness against the newly emerging IAVs and the HPAI viruses. Thus, it is essential to develop novel anti-IAV drugs with new targets. A number of protein-based or small molecule anti-IAV agents have been shown to interfere with the HA-mediated membrane fusion by targeting the receptor binding, blocking the cleavage of HA0, or by inhibiting the low pH-mediated conformation changes of HA. It seems not easy to find or design small compounds targeting the binding event of influenza virus, but the conformational change of HA2 which mediates membrane fusion, is a promising target for developing anti-influenza drugs. Novel influenza virus entry inhibitors may provide more selections for combination therapy with NA inhibitors and M2 ion channel blockers for treating and preventing influenza virus infection and potential pandemic outbreak.
In addition to above inhibitors, some natural molecules also possess good inhibitory activity against influenza A virus infection (Figure 6). For example, catechins isolated from green tea, like EGCG, were found to exhibit mild anti-influenza effect. Further modifications of catechins derived a set of better inhibitors. To be better anti-influenza drug candidates, catechin derivatives possess broad spectrum anti-influenza activity. Besides, curcumin, the widely used spice and coloring agent in Indian food, was proved to be a good virus entry inhibitor targeting HA with EC50 value of 0.47 μM. Modification of curcumin may produce a series of novel HA targeting inhibitors. Another kind of small molecule inhibitor is derived from andrographolide, like AL-1. AL-1 showed significant activity against avian influenza A (H9N2 and H5N1) and human influenza A H1N1 viruses in vitro. AL-1 is capable of direct interfering with viral HA to block viral binding to cellular receptors as was demonstrated by its inhibitory activity on viral adsorption to red blood cells. In 2009, Li’s group in China reported the first three small saponins molecule which inhibit HA. These three compounds can potently inhibit the entry of a H5N1 virus (A/Viet Nam/1203/2004) with IC50at low μM level. Further modifications revealed that the reduction of R1 to a disaccharide chain would abolish the inhibitory activity of these inhibitors. Therefore, anti-influenza agents from natural products, especially those from Traditional Chinese Medicine (TCM), are promising lead compounds. Some lead compounds from TCM are extensively and intensively reviewed by Xu et al. in 2010.
Personal protective equipment is important to prevent transmission of novel A/H1N1 as stated earlier by Shine et al. However, vaccination is the most effective means of preventing influenza transmission and associated morbidity and mortality. It is most important to realize that an effective measure against a pandemic is to have vaccinated and well-informed health care workers.
Unfortunately, the A/H1N1 vaccination coverage was extremely affected by an ongoing public discussion about potential side effects. Therefore, we analysed self reporting questionnaires concerning adverse reactions in 4337 HCWs and medical students.
Of course, this study - that was initiated in the acute event of a pandemic and a safety discussion - has a variety of limitations. Apart from the paucity of demographic data, self reporting questionnaires are largely limited since there may be a number of individuals who do not return the questionnaire despite adverse reaction manifestation. However, there was a need to assess potential adverse reactions since the general population and the HCWs asked for data about the new vaccine. Therefore, we decided to undertake a self reporting study despite that fact that the extent of underreporting of side effects can not be examined precisely in the chosen design. It is noteworthy that with our self reporting system we found a rate of 6.7% (291 of 4337 vaccinations). This frequency differs slightly from data from other studies but points to a safe vaccine in terms of acute adverse reactions. A prospective, randomised study with 178 participants by Vajo et al. concluded that all adverse events were rare, mild, and transient. Using the vaccine Fluval P, the most frequent reactions in this study were pain at injection site (eight cases) and fatigue for 1-2 days after vaccination (three cases). Concerning the vaccination rate we can report a rate of 4337 of about 10 000 employees of the hospital. This is a vaccination rate of over 40%. In a parallel study in Frankfurt/Main, the influenza vaccination rates of the HCWs of the University Hospital Frankfurt were measured. In this study, we were also able to show that the 2009 vaccination rate (seasonal influenza [40.5%], swine flu [36.3%]) was better than the average annual uptake of influenza vaccine in the German health care system (approximately 22% for seasonal and 15% for swine flu).
In meantime, a number of studies were published that also addressed safety issues of the H1N1 vaccination in healthcare workers. I.e. an inactivated, split-virus, unadjuvanted AH1pdm vaccine, manufactured in Japan, was given to HCWs from October 19, 2009. A retrospective cohort study was conducted and and severe adverse events were rare. A recent study using monovalent vaccination (Panenza; Sanofi Pasteur, Val de Reuil Cedex, France) among HCWs in a university hospital setting in Thailand also reported a low rate of side effects. The most common adverse reaction was fatigue/uncomfortable feeling (24%).
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The mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days.
IM was responsible for organization of the study, performance of analyses, data collection and management, data analysis and writing of the manuscript; ME was responsible for statistical analysis and the study design; CL was responsible for coordination of the study, analyses and test performance, RM, LR, AA, RAR, BK, NA, was responsible for patient sample procurement, quality control and assay performance, GP, EM was responsible for the antibody detection assays, AL for epidemiology, NLP for LifeGene’s study design, data analysis and interpretation, MM was responsible for the study design, organization, data analysis, interpretation and writing the manuscript. All authors read and approved the final manuscript.
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.
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This is an immunodiffusion-based approach used to measure antibody responses to influenza virus internal proteins NP and M, following vaccination or infection. Due to lower sensitivity, complement fixation has been replaced by HAI, VN, and EIA assays.
There were several zoonotic influenza strains that infected poultry and human during the past decade in Taiwan (Figure 2). In April 2009, a new strain of H1N1 from Mexico was found to be a novel strain of influenza for which current vaccines against seasonal flu provided little protection. This new H1N1 strain resulted from a reassortment of bird, swine, and human flu viruses. In June 2009, the WHO declared the outbreak of a pandemic which was named as pandemic H1N1/09 virus (pdmH1N1) in July 2009. It is possible that the virus has been circulating in human population since some time in the past and had not been detected.
The first case of H1N1 detected in Taiwan was confirmed on May 20, 2009, and another 9 positive cases in a row were identified within a week. The pdmH1N1 virus was isolated in late May 2009, causing a community outbreak in early July and then spreading islandwide. Taiwan CDC provided free seasonal influenza vaccine started from October; people received their vaccination at over 3,500 contracted hospitals and clinics. Then the Taiwan government approved an inactivated vaccine with influenza A/California/7/2009 (H1N1) strain known as AdimFlu-S (Adimmune Corporation, Taichung, Taiwan) and initiated mass vaccinations in November. Free AdimFlu-S vaccines were provided giving the priority to schoolchildren, the elderly, and front-line healthcare personnel. Due to the delay in vaccine development and delivery in 2010, health authorities used both vaccination and school closure to control pdmH1N1 [22–24]. Overall, the vaccine coverage rates were 76.9% for children and 24.6% for civilians in late July 2010 [22, 24, 25].
Confirmed case of pdmH1N1 influenza virus infection in Taiwan is defined as an individual with laboratory-confirmed pdmH1N1 influenza virus infection by one or more of the following tests: (1) real-time reverse transcriptase-polymerase chain reaction (RT-PCR), viral culture, or fourfold rise in pdmH1N1 influenza virus-specific neutralizing antibodies; (2) viral culture and (3) RT-PCR which can reliably identify the presence of pdmH1N1 influenza virus in specimens, especially RT-PCR, which has the highest sensitivity and specificity. With RT-PCR, pdmH1N1 influenza virus will test positive for influenza A and negative for seasonal H1 or H3. Meanwhile, commercially available rapid influenza diagnostic tests (RIDTs) detect influenza viral nucleoprotein antigen and are capable of providing results within 30 minutes. The sensitivity of RIDTs for detecting pdmH1N1 influenza varied from 10% to 70% and is directly related to the amount of virus in the specimen but inversely related to the threshold cycle value of the test. Rapid tests for influenza may detect the antigen from either influenza A or B in respiratory specimens with a high specificity (>95%), but the negative result from a rapid test does not rule out influenza infection, and most of rapid tests cannot distinguish pdmH1N1 from H3N2 influenza A viruses.
The oseltamivir-resistant pandemic influenza A (H1N1) virus strain in Taiwan was first isolated from a 20-year-old male in October 2009. The H1N1 virus isolated before the patient received oseltamivir treatment was sensitive to oseltamivir. However, three days after initiation of treatment, the virus isolated from the same patient shows a mutation involving the substitution of a histidine for tyrosine at position 275 (H275Y), which is resistant to oseltamivir. Oseltamivir treatment was not associated with statistically significant reduction in the duration of viral shedding, thereby proving ineffective in preventing viral spread in community. The role of swine in the genesis of this pandemic was again apparent. The lesson learned is that the pandemics of influenza can arise anywhere in the world and that global surveillance is merited.
This study reported a high prevalence of respiratory viruses in children ≤ 5 years using a custom assay and an FTD assay. Good concordance was observed for all the viruses between both assays except for RSVA/B. However larger numbers of positive samples need to be tested for thorough evaluation of less prevalent viruses. The custom primer and probe mix was much more economical than the commercial FTD kit. Our study suggests that this custom multiplex real-time RT-PCR can be used for simultaneous and rapid detection of multiple viruses in resource limited settings. This will help prevent unnecessary use of antibiotics and permit timely initiation of supportive therapy/antiviral drugs if available.
In summary, our results suggest that the clinical features correlated to different FLU viruses and to the relevant subtypes should be taken into consideration by health authorities to implement prevention strategies with the aim to reduce the number of sick subjects, the prevalence of hospitalization, and the circulation of FLU.
This research study did not involve any health-related patient interventions. The study was conducted in accordance with the principles and guidelines expressed in the Declaration of Helsinki and was approved as less than minimal risk research by the Research Ethics Committee at PKUPH. Written consent forms were not required and approved information sheets were used instead of consent forms. Detailed information was given to all patients, and once informed consent had been received, swab samples were collected and the data analyzed anonymously.
Promptly after the SARS epidemic, the Chinese government accelerated the establishment of an effectual and national unified management system for public health emergencies and enacted two laws: the Regulation on Public Health Emergency and the Measures for the Administration of Information Reporting on Monitoring Public Health Emergencies and Epidemic Situation of Infectious Diseases [18, 19]. In addition to defining the standards and grades of public health emergencies, these laws support the construction of command systems and clarify the responsibilities and the leadership role of the chief executive of central and local governments previously held by the Centers for Disease Control and Prevention (CDC). Accordingly, the executive capacity of the command systems has been much improved. Importantly, China also established an emergency information dissemination system to enable timely (within 2 hours), accurate, and comprehensive release of information. Moreover, both central and local governments are now expected to be prepared for a public health emergency response (e.g., techniques, personnel, materials, and management preparedness).
In 2004, China revised the Prevention and Treatment of Infectious Diseases Law, adding SARS and avian flu as notifiable diseases and revising the law to comply with the principal rules of infectious disease prevention and control (i.e., infection source control, interruption of route of transmission, and susceptible people protection). The law adheres to the “five early” principle of early detection, diagnosis, reporting, isolation, and treatment. Early isolation can restrain contagion. The law applied China's experience in emerging epidemics to prevent and control 37 infectious diseases.
The Chinese Ministry of Health (CMH) issued a technical guide for avian flu prevention and control in 2004. It requires that suspected and confirmed cases be handled quickly at designated hospitals with the equipment to prevent nosocomial infection. The epidemiological and etiological data of patients should be acquired to enable determination of human-to-human transmission capacity. The guide suggests that persons exposed to dead poultry infected by avian flu virus be isolated and observed for 7 days. In order to control zoonotic infectious diseases, China revised its Law on Animal Disease Prevention in 2007, adding an animal epidemic surveillance and reporting system for timely disclosure of animal epidemics and providing compensation to farmers for economic loss due to culling infected or potentially infected poultry.
Guided by the aforementioned laws, a series of social innovations enacted after the SARS epidemic have improved China's ability to combat emerging diseases. The CMH issued a swine flu prevention guide on April 29, 2009, 12 days before the first reported H1N1 case. On April 3, 2013, 4 days after the first H7N9 confirmed case, the CMH also issued a nosocomial H7N9-infection prevention guide. Administrative reforms resulted in better handling of H5N1, H1N1, and H7N9 relative to SARS. Importantly, in keeping with its move toward greater transparency, after confirmation of the first H1N1 case on May 11, 2009, China posted patient zero's travel information publicly on the same day. Likewise, after confirming the first H7N9 avian flu case on March 30, 2013, China published detailed information about the patient's medical consultation. Transparency helps to subdue rumors and maintain social stability.
Influenza, also known as the flu, is a respiratory illness caused by viruses belonging to the family Orthomyxoviridae. This family consists of four influenza virus genera (influenza virus A, influenza virus B, influenza virus C, and influenza virus D) that are classified based on differences in their internal glycoproteins nucleoprotein (NP) and matrix (M). Influenza type A viruses can infect humans, birds, pigs, horses, and other animals, while influenza B and C viruses are found only in humans. Influenza viruses contain a single stranded negative sense RNA genome that encodes 11 proteins. Based on the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are divided into various subtypes. There are 18 HA (H1–H18) and 11 NA (N1–N11) subtypes of influenza A viruses, that potentially form 144 HA and NA combinations. Aquatic birds including ducks, geese, and swans, are considered to be the natural reservoir of these subtypes.
Each year influenza viruses, both influenza A and influenza B are responsible for seasonal epidemics accounting for over 200,000 hospitalizations and 30,000–50,000 deaths. As per World Health Organization (WHO) estimates, influenza viruses infect between 5%–15% of the global population, annually resulting in 250,000 to 500,000 deaths, making it the leading cause of mortality after acquired immune deficiency syndrome (AIDS). In addition to annual seasonal epidemics H1N1 and H3N2 viruses have also resulted in four major influenza pandemics: The “Spanish flu” in 1918, the “Asian flu” in 1958, the “Hong Kong flu” in 1968, and the more recent 2009 H1N1 pandemic. Since 1997, human infections with a novel H5N1 subtype of highly pathogenic avian influenza (HPAI) have been reported. The first cases of human infection with H5N1 influenza were reported in 1997, when HPAI outbreaks in poultry farms and markets in Hong Kong resulted in eighteen cases and six deaths. Since then, this virus has spread to many countries in Asia, Africa, and Europe, resulting in over 424 human cases with a mortality rate greater than 60%. In addition, H9N2 and H7N7 avian influenza subtypes have also been reported to cause human infections. The most recent strain infecting humans was H7N9 in China, detected in 2011.
A number of diagnostic techniques, including virus isolation, nucleic acid amplification test (NAAT), immunochromatography-based rapid diagnostic test (RDT), etc., have been used for detection of influenza viruses in humans. Here, we review various approaches currently available or under development for diagnosis of influenza infections in humans.
The meeting participants agreed that assessing the burden of disease and the impact of vaccination is more difficult for influenza than for other vaccinepreventable diseases because influenza cannot be eradicated, symptoms are nonspecific, cases are not usually virologically tested, and herd protection is difficult to measure.
Immunization rates are decreasing in Argentina and Brazil (9, 15) for flu as well as other vaccines, although both countries still retain the highest influenza vaccination rates worldwide. One reason for this downward trend is parent misinformation. Parents often see influenza as a mild disease and thus view the influenza-like symptoms from the vaccines as outweighing any benefits (29). In addition, people get tired of having to get shots every year. The growing influence of local anti-vaccination advocacy groups is also a cause for concern.
Another factor in the lower coverage is the logistics related to limited staffing for administering routine vaccines in a crowded childhood immunization calendar.
Education of health care workers is another problem that needs to be tackled. As shown in a study evaluating missed opportunities for flu vaccination, from the parents’ perspective (30), the main reason for the lower coverage was a lack of information from health care workers, who were not recommending the vaccine. In Argentina, few specialists recommend influenza vaccination for children with chronic comorbidities at highrisk for flu-related complications.
In discussing the best vaccine options, the meeting participants emphasized the superiority of the aIIV3 compared to the IIV4. While the second B strain may add 15% efficacy, leading to an overall IIV4 efficacy of 60%–65%, the efficacy of adjuvanted vaccines exceeds 80%, while also providing protection for mismatched B lineage. In Brazil, although the IIV4 became available in the private market in 2015, the Ministry of Health is not considering incorporating it into the NIP in the near future.
Safety concerns for use of repetitive doses of adjuvanted vaccines in young children were also addressed. Canada has licensed the aIIV3 with limited indication in the youngest age group (6–24 months) (26). In Argentina, the national immunization committee has resolved to continue using IIV3 and will consider incorporating adjuvanted vaccines in the future (a technology transfer agreement will enable local vaccine production).
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).
Only participants with confirmed H1N1 by qRT-PCR and ILI of any age who had a fever ≥38°C, cough, and headache accompanied by one or more of rhinorrhea, rhinitis, arthralgia, myalgia, prostration, sore throat, chest pain, abdominal pain, or nasal congestion were included in the study. For patients <5 years of age, irritability was substituted for headache. In those >65 years of age, fever was not required as a cardinal symptom.
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The presence of all subtypes influenza A viruses in wild aquatic birds poses serious health risks to a wide range of animal species. Influenza A viruses are enveloped, single-, negative-stranded and segmented RNA viruses belonging to the Orthomyxoviridae family; they are highly infectious respiratory pathogens in their respective natural hosts. All 16 known hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A viruses have been isolated from wild waterfowl and seabirds (Webster et al, 2006). Although some of these subtypes are non-pathogenic/non-virulent within their natural hosts and have been present in these animal reservoirs for many centuries, various subtypes are highly virulent within their natural host species and to other species (Webby et al, 2007). For example, the changing role of the highly pathogenic avian influenza virus (HPAIV) H5N1 subtype in both wild and domestic ducks has recently been documented as a potential public health hazard because they are zoonotic agents with the theoretical ability – after genetic adaptation – of a human-to-human transmission, i.e., the start of a human epidemic/pandemic (Hulse-Post et al, 2005).
The ecology of influenza A viruses is dynamic and complex involving multiple host species and viral genes. Commercial poultry farms, “wet markets,” (where live birds and other animals are sold), backyard poultry farms, commercial and family poultry slaughtering facilities, swine farms, human dietary habits and the global trade in exotic animals have all been implicated in the spread of influenza A viruses (Greger, 2006). The “wet markets” of Southeast Asia, where people, pigs, ducks, geese and chickens (and occasionally other animals) are in close proximity pose a particular danger to public health (Webster, 2004; Bush, 2005; Greenfeld, 2006; Lau et al, 2007).
Scientific opinion differs on the probability of future sustained human-to-human transmission, e.g., for H5N1 HPAIV, as well as on which viruses pose the greatest threat to humanity and to other species (Hilleman, 2002). The prevailing scientific view regarding a possible H5N1 epidemic is that sustained human-to-human transmission will occur at some unknown future date and that a prediction on the future virulence of H5N1 viruses to humans is very difficult to make. The H5N1 HPAIVs are possibly the greatest threat at the moment, although H1, H2, H3, H7 and H9 avian-derived viruses are also strong contenders as causes of potential epidemics in various species, including humans (Hilleman, 2002; Wan et al, 2008). This review will conclude that an avian influenza virus transmitted via pigs to humans poses a significant risk to cause a new influenza pandemic, possibly on the disturbing scale of the human influenza pandemic experienced during 1918-1920 (H1N1 “Spanish Flu”). Before investigating the role of swine in the influenza A viruses, it is necessary to consider the challenge which influenza A viruses pose to both human and animal health.
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.
Influenza viruses A and B are the main pathogens responsible for the onset of epidemics because of their evolving nature. They are RNA viruses that have a high mutation rate and ability to make “drift” changes; however, only influenza A viruses are responsible for pandemics. Worldwide, influenza A viruses are the cause of severe infections in 3–5 million people annually, and these viral infections kills 0.25–0.5 million people annually.44 As such, influenza outbreaks produce high morbidity and mortality rates with great economic and social impact.44
Early findings in relation to the most recent influenza pandemic occurred in April 2009 in Mexico and soon spread to other countries. The pandemic was caused by an H1N1 variant, which came from two genetic recombination events. The first occurred in 1998, when an avian virus, an American pig virus, and virus fragments of humans had exchanged genetic materials. The following recombination with a European swine virus strain resulted in the pandemic swine origin influenza virus.2,30 In Mexico in recent years, this has caused at least four outbreaks with high mortality rates compared with that presenting in other countries.6 During the winter of 2017–2018, influenza activity increased in Mexico, and 2,855 cases of influenza and 73 deaths were confirmed by March 02, 2018, of which 46 cases were A(H3N2), 11 cases A(H1N1) pdm09, 10 cases B, and the remaining six cases were not subtyped.39
Results emanating from different studies have shown that influenza outbreaks are characterized by high severity of symptoms with increased mortality,6,8,13,25,32 Several factors have been associated with H1N1 disease severity, such as factors due to the virus (ie, viral pathogenic mutations, resistance to antivirals), factors inherent to host susceptibility (eg, age, sex, race), including physiological immunosuppression or acquired diseases (ie, diabetes, hypertension, obesity, asthma), factors associated with available medical services and public health facilities, and factors arising from the presence of bacterial coinfections.1,3,6,8,9,16,18,25,32,34,35,41
Seasonal and pandemic influenza often have complications arising from bacterial coinfections. Cillóniz et al12 documented that in H1N1 patients with community-acquired pneumonia, the most frequently isolated bacterial pathogens were Streptococcus pneumoniae (26, 62%) and Pseudomo-nas aeruginosa (6, 14%). Staphylococcus aureus was rarely found, and Haemophilus influenzae was not found.12 During the 1918 pandemic, most deaths had bacterial coinfections. Globally, more than 34% of influenza virus infections needed intensive care among hospitalized patients, from which 0.5% of all cases of influenza corresponded to healthy young individuals and at least 2.5% of total cases the elderly group and those with coinfections harbored the bacteria.27 Symptoms of influenza cases with bacterial coinfections are similar to those with severe influenza, but the former may have a higher risk of death. Identification of coinfections should be considered in patients with influenza-like illness (ILI) presenting symptoms suggestive of pneumonia, such as dyspnea, tachypnea, and hypoxia, or with evidence of septicemia.27 Many copathogens are known to be colonizers of the respiratory mucosa, ie, S. pneumoniae, H. influenzae, and Neisseria meningitidis, including the upper and lower respiratory tracts. Distinction between copathogenic colonization and coinfection is critical, because a proven coinfection is clinically correlated with signs of pneumonia and bacterial contagion producing increased acuity or severity of disease.27
Empirical antiviral treatment should be considered and managed in such critically ill patients. The most commonly isolated bacterial pathogens are those that colonize the nasopharynx, and this complex of virus–bacteria contributes significantly to the pathogenesis of the disease, mainly in periods of endemic influenza.26,34 There have been studies reporting copathogens between influenza and other viruses, but few cases have observed that this produced severe complications because of coinfection.15,33,42 Two studies have hypothesized a “viral interference”, suggesting that a rhinovirus infection may interfere with the A(H1N1pdm09) influenza, but this is still not fully understood.24,33
The precise identification of infectious pathogens responsible for acute respiratory infections, primarily influenza, is a critical factor for proper treatment of the disease and control during outbreaks and for the appropriate use of antibiotics and antivirals. For these reasons, continuation of investigations into the pathogens commonly associated with influenza cases is urgently needed. Here, the presence of bacterial and viral copathogens are identified using clinical samples for the molecular diagnosis by resequencing microarray in study patients with confirmed influenza A(H1N1)pdm09 in Oaxaca, Mexico. We also document an association between influenza A(H1N1pdm 09) and symptoms of disease severity in dead patients with multiple-microbial infection.
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.
Raina MacIntyre receives funding from influenza vaccine manufacturers GSK and CSL Biotherapies for investigator-driven research. Kirsten Ward has received funding from vaccine manufacturer Wyeth to attend a conference. Neither of these payments was associated with this study. The remaining authors have no competing interests.