Molecular Determinants of Drug Resistance and Mutation Patterns in Influenza Viruses Circulating in Poland Across Multiple Epidemic Seasons: Implications for Vaccination Strategies.

BACKGROUND According to the WHO, up to 650 000 people die each year from seasonal flu-related respiratory illnesses. The most effective method of fighting the virus is seasonal vaccination. However, if an infection does occur, antiviral medications should be used as soon as possible. No studies of drug resistance in influenza viruses circulating in Poland have been systematically conducted. Therefore, the aim of the present study was to investigate the drug resistance and genetic diversity of influenza virus strains circulating in Poland by determining the presence of mutations in the neuraminidase gene. MATERIAL AND METHODS A total of 258 clinical specimens were collected during the 2016-2017, 2017-2018, and 2018-2019 epidemic seasons. The samples containing influenza A and B were analyzed by RT-PCR and Sanger sequencing. RESULTS Differences were found between the influenza virus strains detected in different epidemic seasons, demonstrating the occurrence of mutations. Influenza A virus was found to be more genetically variable than influenza B virus (P<0.001, Kruskal-Wallis test). However, there was no significant difference in the resistance prevalence between the influenza A subtypes A/H1N1/pdm09 (4.8%) and A/H3N2/ (6.1%). In contrast, more mutations of drug-resistance genes were found in the influenza B virus (P<0.001, chi-square test). In addition, resistance mutations appeared en masse in vaccine strains circulating in unvaccinated populations. CONCLUSIONS It seems important to determine whether the influenza virus strains tested for drug resistance as part of global influenza surveillance are equally representative of viruses circulating in populations with high and low vaccination rates, for all countries. Our results suggest that countries with low levels of influenza immunization may constitute reservoirs of drug-resistant influenza viruses.

Treatment and prophylaxis of influenza and the problem of resistance to neuraminidase inhibitors.

Influenza virus neuraminidase inhibitors (NAIs), including oseltamivir, zanamivir and peramivir, are important antivirals for the treatment and prophylaxis of influenza. Increasing use of NAIs brings into focus the risk of drug resistance. The problem of resistance is of high clinical and epidemiological importance. There are generally three levels of antiviral resistance according to the way that resistance can be detected or inferred: genotypic, phenotypic and clinical resistance. Recently the problem of resistance to NAIs, although still rare (<2% of influenza isolates), has been rising. It should be underlined that NAI resistance in influenza viruses is relative, and despite its presence patients with resistant viruses may still benefit from receiving NAIs. The clinical resistance and the response to treatment with antivirals remain the most important proof of antiviral effectiveness. Currently, there has not been observed cross-resistance between oseltamivir and zanamivir, which may be a consequence of the number of given doses, differences in drug structure and duration of the drug concentrations in the site of infection. Early treatment with appropriate doses of NAI is necessary to minimize the likelihood of a resistant virus arising.

Prevention of influenza infection--a Polish perspective.

Influenza is a viral respiratory illness that causes high morbidity and significant mortality in humans. Costs associated with influenza in terms of human suffering are immeasurable and the economic costs are very high. Every year, according to the World Health Organization (WHO), 5-25% of the global population suffers from infection with influenza and influenza-like viruses and between 500 thousand and one million individuals of all ages die from multiple organ complications, irrespective of the geographic location. Influenza vaccination is still neglected and the percentage of the global population vaccinated remains low. The first authorization for the use of influenza vaccines in humans was issued in 1941. Currently, many varieties of influenza vaccine are available, containing either fragments of inactivated influenza virus or live vaccine which consists of attenuated virus. The influenza vaccine is most often developed in chick embryos or less frequently in tissue culture such as MDCK and Vero. A variety of inactivated vaccines are registered in Poland. Due to the mutability of the virus, it is not yet possible to develop a universal vaccine, nor can the disease be eradicated; however, prevention is possible by inoculating the greatest percentage of the global population. According to the WHO, Poland is in the penultimate position in Europe in terms of the percentage of the population vaccinated. In the last epidemic season of 2012/2013 only 3.75% of the Polish population was immunized.

[Influenza vaccinations of health care personnel]. / Szczepienia przeciwko grypie u personelu medycznego.

Influenza is one of the most common respiratory diseases affecting people of all age groups all over the world. Seasonal influenza leads to substantial morbidity and mortality on a global scale. Vaccines are undeniably one of the most important health advances of the past century, however, managing influenza in working populations remains a difficult issue. Vaccination of health care workers (HCW) is an efficient way to reduce the risk of occupational infection and to prevent nosocomial transmission to vulnerable patients. Despite this, achieving high immunization rates among those professionals is a challenge. Knowledge and attitudes of healthcare providers have significant impact on the frequency with which vaccines are offered and accepted, but many HCWs are poorly equipped to make informed recommendations about vaccine merits and risks. Principal reasons for vaccination are the willing not to be infected and avoiding transmission to patients and the family. The main reasons for refusing is lack of time, a feeling of invulnerability to vaccination, conviction of not being at risk, of being too young or in good health. Misconceptions about influenza vaccine efficacy, like adverse effects, and fear of contracting illness from the vaccine are significantly associated with noncompliance with vaccination. Therefore, strategies to increase awareness of the importance of recommending influenza immunization among health professionals are required.

Opinions of employees of the National Institute of Public Health--National Institute of Hygiene in Warsaw on influenza vaccination.

INTRODUCTION: Improving influenza vaccination coverage is an important action to prevent influenza epidemics and reduce the costs caused by the epidemics. Recognising the motives to be vaccinated or failure to vaccinate, especially among health care workers, is needed. OBJECTIVES: The aim of presented papers is: 1) recognising the influenza vaccination coverage among NIPH-NIH employees, 2) examining the determinants of decision be vaccinated/not vaccinated, 3) estimating the effectiveness of influenza vaccination in relation to sickness absence due to respiratory infection. MATERIAL AND METHODS: The study was carried out in NIPH-NIH by e-mail questionnaire. Out of 345 employees, 187 (54,2%) participated in the study. The questionnaire contained information on influenza vaccination and determinants that would potentially affect the decision to vaccinate. RESULTS: 18,7% of the participants was vaccinated in the previous epidemic season and the half of employees were vaccinated at least one time in the previous 10 seasons. Only every fourth family/occupational doctor encouraged their patients to vaccinate. The NIPH-NIH employees would be more likely to be vaccinated, if the employer has provided free vaccines. The estimation of influenza vaccination effectiveness in decreasing the sickness absence due to respiratory infection amounted 37%. CONCLUSIONS: Our findings confirmed that influenza vaccination contributes to noticeable decreasing of sickness absence. Providing free vaccination against influenza by employer could increase considerably the coverage.

[Vaccination against influenza in pregnant women - safety and effectiveness]. / Szczepienia przeciwko grypie u kobiet ciezarnych - bezpieczenstwo i efektywnosc.

Influenza is a major cause of morbidity and mortality worldwide. During seasonal influenza epidemics and pandemics, pregnancy places otherwise healthy women at an increased risk of complications from influenza. The factors believed to increase the susceptibility of complicated influenza infection during pregnancy are linked to the physiologic changes, including immunologic changes (attenuation of the cell-mediated immune responses, selective suppression of T-helper 1 cell mediated immunity while the adaptive humoral immunity remains unimpaired), increased cardiac output and oxygen consumption and tidal volume. Pregnant women have similar incidence of seasonal influenza as the general population, however because of the physiological changes, they are at an increased risk of complications (including secondary pneumonia, acute respiratory insufficiency increased risk of stillbirth, premature deliveries) and death. Immunization of pregnant women against influenza is currently recommended in many countries. Vaccination against influenza with trivalent inactivated vaccine (TIV) has been proven to be safe and effective. Lack of harmful effect of TIV on pregnant women and newborns has been demonstrated in several studies: no increased risk of spontaneous abortions, preterm birth, low birth weight, congenital malformations, cesarean section have been reported. Vaccination against influenza has been proven to be effective in reducing rates and severity of the disease in vaccinated mothers and their children. Several studies revealed a decreased risk of influenza-like illnesses among mothers who were vaccinated during pregnancy but also a decreased risk of laboratory confirmed cases of influenza and hospitalizations due to influenza and its complications among newborns and infants born to vaccinated mothers. Currently available inactivated influenza vaccines are not licensed for use in infants younger than 6 months. Protection of young infants against the infection in early life thus requires a cocooning strategy to reduce the number of vulnerable individuals among care givers and contacts. Neonates and infants may be also protected against influenza directly by antibodies of maternal origin that cross the placenta or are transferred via breast milk. The duration of passively acquired antibodies depends on the initial blood concentration and is probably less than 6 months. Vaccine coverage among pregnant women rdmains low Possible explanations include lack of education by health care workers, the feeling among the general public that influenza is not a serious problem, and the failure of prenatal care providers to offer the vaccine. Overall, the most important factor for a woman to decide to be immunized during pregnancy was to have a clear recommendation from the health care provider Reasons evoked by obstetricians for not providing influenza vaccines included lack sufficient data on safety and efficacy concerns about the medical legal risks of vaccination during pregnancy and the perdeption that pregnant women would not want to be vaccinated. Educational intervention targeting health care workers in charge of pregnant women should be primary implemented to provide higher influenza vaccine coverage and to protect pregnant women and young infants from influenza related morbidity

The Impact of the COVID-19 Pandemic on Influenza Transmission in Poland.

BACKGROUND: The aim of this study was to determine whether the occurrence of the SARS-CoV-2 pandemic affected the incidence of influenza in Poland and the efficiency of the SENTINEL influenza surveillance system. METHODS: The analysis was based on virologic data from the 2018/2019-2021/2022 epidemic seasons. The data in question were obtained from the SENTINEL influenza surveillance system, which is utilized in Poland. RESULTS: In the 2020/2021 epidemic season, only one positive case was confirmed. In the epidemic season of 2021/2022, the number of positive cases increased. There was a delay in the peak of the season, since the start of pandemic, which was observed in the 14th week of 2022. Previously, it was recorded in the 5-10th week, depending on the season. Before the pandemic, the number of positive samples in relation to the tested ones oscillated between 41-49.4%. After the pandemic, it was 0.3% and below 20%, respectively, for season 2020/2021 and season 2021/2022. CONCLUSIONS: The COVID-19 pandemic caused a decline in many other infectious diseases, including influenza, as a result of the numerous lockdowns and from people shifting to remote work. Other safety measures, such as obligatory protective masks and the use of disinfectants, had a significant impact on reducing the number of cases.

Accuracy of rapid influenza diagnostic test and immunofluorescence assay compared to real time RT-PCR in children with influenza A(H1N1)pdm09 infection.

INTRODUCTION: The influenza burden among children is underestimated. The aim of our study was to estimate the accuracy of the rapid influenza detection test (RIDT) BD Directigen™ EZ Flu A+B® and direct immunofluorescence assay (DFA) used among children with influenza-like illness (ILI) consulted in the ambulatory care clinic. MATERIAL/METHODS: A total of 150 patients were enrolled in the study. Inclusion criteria were: age less than 59 months, presentation of ILI according to the CDC (Centers for Disease Control and Prevention) definition (fever >37.8°C, cough and/or sore throat in the absence of another known cause of illness), duration of symptoms shorter than 96 hours. Two nasal swabs and one pharyngeal swab were obtained from patients and tested by RIDT, DFA and real time RT-PCR as the reference method. RESULTS: For influenza A (H1N1)pdm09 virus sensitivity of RIDT was 62.2% (95% CI 46.5-76.2%), specificity 97.1% (95% CI 91.8-99.4%), PPV 90.3% (95% CI 74.3-98%), NPV 85.7% (95% CI 78.1-91.5%), for DFA sensitivity was 60% (95% CI 51.9-63.2%), specificity 96% (95% CI 88.7-98.8%), PPV 93.1% (95% CI 80.5-98%), NPV 72.7% (95% CI 67.2-74.9%). Analysis of logistic regression revealed that the chance of receiving a true positive result of RIDT was twice as high when the test was conducted during the first 48 hours of symptoms (OR 0.40 vs OR 0.22). CONCLUSIONS: The accuracy of RIDT is comparable with DFA and both methods are very specific but moderately sensitive in diagnosis of influenza in young children. Both methods may be recommended for screening for influenza among children.

Hemagglutination inhibition antibody titers as a correlate of protection against influenza disease in the 2018/2019 epidemic season in Poland.

The aim of this study was to determine the level of antibodies against hemagglutinin of influenza viruses in the sera of people in the seven age groups in the epidemic season 2018/2019 in Poland. The level of anti-hemagglutinin antibodies was determined by hemagglutination inhibition test (HAI). 1050 clinical samples from all over the country were tested. The level of antibodies against influenza viruses was highest in the 10-14 age group for A/Singapore/INFIMH-16-0019/2016 (H3N2) and B/Phuket/3073/2013 Yamagata lineage antigens. These results confirm the circulation of four antigenically different influenza virus strains, two subtypes of influenza A virus - A/Michigan/45/2015 (H1N1)pdm09 and A/Singapore/INFIMH-16-0019/2016 (H3N2) and two lineages of influenza B virus - B/Colorado/06/2017 - Victoria lineage and B/Phuket/3073/2013 Yamagata lineage.

[Influenza and influenza-like illnesses in Slaskie voivodship in 2000-2005]. / Grypa i zachorowania grypopodobne w województwie slqskim w latach 2000-2005.

UNLABELLED: Influenza is a seasonal viral disease. In Poland several thousand to several million cases and suspected cases of influenza are registered every epidemic season. The active participation of all 16 voivodships in SENTINEL the integrated epidemiological and virological surveillance of influenza and influenza-like illnesses, as well as the increase in flu vaccinations in each voivodship, is of significant importance, not only on account of seasonal morbidity, but also of a possible influenza pandemic. The aim of the study was to analyse influenza and influenza-like illnesses, taking into consideration incidence, hospitalisation and mortality, as well as isolation of the influenza virus and vaccination against flu in the Silesian voivodship in the years 2000-2005. MATERIALS AND METHODS: The analysis was carried out on the basis of annual reports concerning influenza and influenza-like illnesses in districts in the Silesian voivodship between 2000 and 2005. Results. The highest incidences of influenza and influenza-like illnesses in the Silesian voivodship in the years 2000-2005 were registered in the following districts: Wodzislaw Slaski in 2000 and 2003, Jaworzno in 2001 and 2005, and Lubliniec in 2002, while the lowest incidence was noted in the Wodzislaw slaski district in 2004. The peak of incidence of influenza and influenza-like illnesses was observed between January and March of a given year. The percentage of people vaccinated against flu in the above voivodship compared with the number of people vaccinated in Poland was between 10.7 and 12.8. CONCLUSIONS: From an analysis of the period 2000-2005, we can conclude that, besides epidemiological surveillance, it is essential to introduce virological surveillance. Lack of pathognostic symptoms makes it difficult to present a diagnosis merely on the basis of a clinical case definition. Laboratory tests have a key role in diagnosing laboratory infections. Systematic health education is essential in Poland to popularise vaccinations against flu. This is particularly important in view of the fact that only about 8% of the Polish population is vaccinated every season. In EU countries the number of vaccinations is 3 times greater.

[Various sides of influenza, part I--structure, replication, changeability of influenza viruses, clinical course of the disease, immunological response and laboratory diagnostics]. / Grypa w róznych aspektach. Czqsc I--Budowa, replikacja i zmiennosc wirusów grypy oraz przebieg kliniczny choroby, odpowiedz immunologiczna i diagnostyka laboratoryjna.

Influenza viruses represent Orthomyxoviridae family. Spherical virions are 80-120 nm in diameter and have two-layer lipid envelope. The following proteins are coded by 8 or 7 segments of the single-stranded RNA: nucleoprotein (NP), polymerase PB2, PB1 and PA, member protein--M1 and M2, glycoproteins--hemagglutinin (HA) and neuraminidase (NA). HA and NA form spikes on the virion surface. On the basis of antigenic differences there are distinguished three types of influenza virus-A, B and C. Besides, influenza A viruses occur in different subtypes, depending on the features of HA and NA. One of influenza characteristics is its antigenic changeability: antigenic drift and antigenic shift. Infection occurs by droplet route, sometimes through direct contact with infected person or surface. Influenza virus attacks epithelial cells of upper respiratory tract, where replication takes place resulting in the production of approximately 1000 of progeny virions during a single 6-12 h cycle in one cell. Necrosis of ciliary cells of mucosa facilitates invasion of bacterial pathogens. Incubation period lasts on average 1-2 days. Influenza illness without complications characterizes the sudden onset of respiratory symptoms and systemic symptoms. Regression of symptoms usually occurs after 3-5 days, but cough and malaise may be observed for over 2 weeks. Reasons for the severe course of the disease or even death are post-influenza complications, e.g. viral pneumonia and bronchitis, bronchiolitis in children, secondary bacterial pneumonia, otitis media, myocarditis and pericarditis, Reye's syndrome, myositis, myoglobinuria, neurological complications and exacerbation of existing chronic diseases. In the case of influenza there is no possible to make the unquestionable diagnosis only on the basis of clinical picture of the disease. Therefore in some circumstances there is important to make some diagnostic laboratory tests as RT-PCR, immunofluorescence assay or isolation of virus and detection of the specific antibodies. The main determinants of the immunity to influenza virus infection are antihemagglutinin (anti-HA) antibodies and antineuraminidase antibodies (anti-NA). The former play fundamental role for the protection against the infection, while anti-NA antibodies limit virus spreading and contribute to a milder course of the disease. In the response to influenza infection there are observed serum immunoglobulines IgG and IgM (after the first contact with the antigen), while immunoglobulines IgA are produced rarely. The latter are produced locally in the high concentrations on the mucus of respiratory tract. Cellular immunological response is important for recovery from influenza where a significant role of cytotoxic T lymphocytes should be emphasized. These lymphocytes are able to kill infected cells in the earliest phases of replication before the progeny virions are formed.

[Various sides of influenza. Part II--epidemiology, influenza surveillance and prophylaxis]. / Grypa w róznych aspektach. Czqsc II--Epidemiologia i nadzór nad grypq oraz profilaktyka.

Influenza viruses cause epidemics and pandemics. Animal reservoir for influenza viruses and repeatedly occurring human infections with avian influenza viruses since 1997 indicate that there is a real threat of pandemic, but it is no possible to predict when this outbreak begins. Every year 330-990 millions of people are ill due to influenza worldwide. In Poland there are registered from a few hundred to few millions of cases and suspected cases of influenza each epidemic season. WHO coordinates the international Global Influenza Surveillance Network consisting of international WHO reference centres for influenza in London, Atlanta, Tokyo, Melbourne and 115 national influenza centres in 86 countries all over the world. One of such National Influenza Centers is located in Poland at the National Institute of Hygiene in Warsaw Influenza surveillance provides information on the currently circulating strains of influenza viruses, including new variants and their spreading that allows to choose appropriate strains to include into the vaccine for the next epidemic season. Regional influenza surveillance networks also exist, as the European Influenza Surveillance Scheme (EISS). The epidemic season 2004/2005 was the first season when the integrated system of virological and epidemiological surveillance SENTINEL started to work in Poland, according to EISS indications. In this system there are included the selected family physicians representing the entire country, Voivodship Sanitary-Epidemiological Stations and the National Influenza Center. Data obtained by the SENTINEL system are then forwarded to EISS and WHO. Widely available way of prophylaxis against influenza is vaccination. Inactivated influenza vaccines prevent illness in 70%-90% healthy adults under 65 and in children. They also prevent post-influenza complications. Attenuated nasal influenza vaccines are registered to use in Russia and the United States. In Poland, vaccinations against influenza are considered in the Program of the Preventive Vaccinations as recommended since 1994. Vaccination against influenza is especially recommended for people who highly risk occurrence of post-influenza complications as well as for some groups of people due to epidemiological indications. Vaccine is administered before epidemic season, but it is also possible during the epidemic season when virus already circulates in the population. There are performed studies to improve currently vaccines, e.g. studies on the safe adjuvants (aluminium, MF59, virosoms), DNA vaccines, use of hemagglutinins and neuraminidase obtained by genetic recombination and expression in insect cells, studies on the production of influenza vaccines in the cell culture MDCK and Vero instead of currently used chicken embryos. There are available specific antiviral-antiinfluenza drugs of new generation: oseltamivir and zanamivir that are neuraminidase inhibitors and old generation drugs: amantadine and rimantadine that are inhibitors of M2 protein.

Vaccination against influenza in pregnant women.

Pregnancy places otherwise healthy women at an increased risk of complications arising from an influenza infection. It is suggested that physiological changes such as immunological changes, increased cardiac output and oxygen consumption, as well as lung tidal volume might increase the susceptibility to influenza complications if infection occurs during pregnancy. Immunization of pregnant women against influenza is currently recommended in many countries and has been proven to be safe and effective in reducing rates and severity of the disease in vaccinated mothers and their children. Influenza vaccination is also cost-effective. Nevertheless, influenza vaccine coverage remains low in pregnant women. This might stem from the lack of healthcare workers' education, a feeling among the general public that influenza is not a serious disease and a failure of prenatal care providers to offer the vaccine. In order to protect pregnant women and infants from influenza related morbidity and mortality an educational programme targeting healthcare workers in charge of pregnant women should be implemented.

Influenza viruses resistant to neuraminidase inhibitors.

Neuraminidase inhibitors (NAIs) are antiviral drugs for treatment and prophylaxis of influenza. By blocking the activity of the enzyme neuraminidase, NAIs prevent new viral particles from being released. The increasing use of NAIs brings into focus the risk of drug resistance arising to the class. There are three levels of antiviral resistance according to the way that resistance can be detected or inferred: genotypic, phenotypic and clinical resistance. For many years seasonal influenza viruses resistance to NAIs was low (0.33%). Recently, there has been described an increasing number of resistant seasonal influenza strains to oseltamivir (2% in adults, 5-18% in children). In 2007 there were published data describing 14% resistant to oseltamivir strains of influenza A/H1N1/ in Europe. Approximately 0.5-1.0% of influenza A/H1N1/pdm09 isolates are currently resistant to oseltamivir. The established markers of the resistance to oseltamivir were found in 2.4% of human and 0.8% of avian isolates of influenza A/H5N1/. It has been not observed a cross resistance among oseltamivir and zanamivir. NAIs resistance in influenza viruses is relative and despite its presence patients with resistant viruses may still benefit from receiving these antivirals. The response to treatment with antivirals remains the most important proof of antiviral effectiveness. The rational use of NAIs is essential to preserve the best choice for treatment and prophylaxis of seasonal, avian and pandemic influenza.

[Influenza and flu prophylaxis--a current medical issue]. / Grypa i jej profilaktyka--ciagle aktualny problem medyczny.

Influenza is a disease which returns every epidemic season in different degrees of severity, especially in high-risk groups. According to current data, 5-25% of the world population contract this disease and mortality because of influenza reaches 0.5-1 million people. However, this problem is still not dealt with appropriately and is even disregarded. In contrast to other viruses influenza cannot be eradicatedas a result of its changeability but post-influenza complications can be dealt with using prophylactic methods. Since 9 May 1977 there have been more and more fatal cases of human infections with bird flu. The danger of another outburst of a flu pandemic is imminent. According to World Health Organisation influenza is one of the priorities of public health--that is the reason of originating International Influenza Surveillance Programme. A basic method of protecting the population against influenza, which is also the cheapest, is vaccination of as many people in the population as possible, especially those from high-risk groups. Post-influenza complications result not only in illness but also in economic losses.