Oropharyngeal Microbiome Analysis in Patients with Varying SARS-CoV-2 Infection Severity: A Prospective Cohort Study.

Patients with COVID-19 infection have distinct oropharyngeal microbiota composition and diversity metrics according to disease severity. However, these findings are not consistent across the literature. We conducted a multicenter, prospective study in patients with COVID-19 requiring outpatient versus inpatient management to explore the microbial abundance of taxa at the phylum, family, genus, and species level, and we utilized alpha and beta diversity indices to further describe our findings. We collected oropharyngeal washing specimens at the time of study entry, which coincided with the COVID-19 diagnosis, to conduct all analyses. We included 43 patients in the study, of whom 16 were managed as outpatients and 27 required hospitalization. Proteobacteria, Actinobacteria, Bacteroidetes, Saccharibacteria TM7, Fusobacteria, and Spirochaetes were the most abundant phyla among patients, while 61 different families were detected, of which the Streptococcaceae and Staphylococcaceae families were the most predominant. A total of 132 microbial genera were detected, with Streptococcus being the predominant genus in outpatients, in contrast to hospitalized patients, in whom the Staphylococcus genus was predominant. LeFSe analysis identified 57 microbial species in the oropharyngeal washings of study participants that could discriminate the severity of symptoms of COVID-19 infections. Alpha diversity analysis did not reveal a difference in the abundance of bacterial species between the groups, but beta diversity analysis established distinct microbial communities between inpatients and outpatients. Our study provides information on the complex association between the oropharyngeal microbiota and SARS-CoV-2 infection. Although our study cannot establish causation, knowledge of specific taxonomic changes with increasing SARS-CoV-2 infection severity can provide us with novel clues for the prognostic classification of COVID-19 patients.

Cellular and Humoral Responses in Dialysis Patients after Vaccination with the BNT162b2 or mRNA-1273 Vaccines.

The outbreak of SARS-CoV-2 has raised considerable concern about the detrimental effects it can induce in public health, with the interest of the scientific community being focused on the development of preventive and therapeutic approaches. Patients with end-stage renal disease (ESRD) are amongst vulnerable populations for critical illness owing to the presence of other comorbidities, their defective immune system, and their inability of self-isolation. To date, vaccination constitutes the most promising method to manage viral dispersion. Therefore, it is particularly important to investigate the effectiveness of available vaccines against SARS-CoV-2 in this risk group. Here, we summarize initial experience regarding the humoral and cellular immune responses elicited in dialysis patients after completion of the recommended vaccination regimen, as well as after booster dose administration, with one of the two mRNA vaccines, namely, BNT162b2 and mRNA-1273. In conclusion, a significantly diminished and delayed immune pattern was observed in ESRD patients compared to healthy population, with a peak in antibody titers occurring 3-5 weeks after the second dose. A booster dose significantly augmented the immune response in dialysis patients with either mRNA-based vaccine. Variables adversely correlating with the weak immunogenicity observed in dialysis patients include immunosuppressive therapy, older age, comorbidities, longer time in hemodialysis treatment, and higher body mass index. On the contrary, previous COVID-19 infection and administration of the mRNA-1273 vaccine are deemed to induce a more favorable immune response. Further investigation is needed to thoroughly understand the efficacy of mRNA-based vaccines in hemodialysis patients and define predictive factors that can influence it.

Clinical Performance Evaluation of the NeuMoDx Flu A-B/RSV/SARS-CoV-2 Vantage Assay.

SARS-CoV-2 infections may present with various symptoms that are similar to those of other respiratory diseases. For this reason, the need for simultaneous detection of at least RSV and influenza viruses together with SARS-CoV-2 was evident from the early stages of the pandemic. In the present study, we evaluated the clinical performance of the NeuMoDx™ Flu A-B/RSV/SARS-CoV-2 Vantage Assay against the conventional low-plex PCR utilized to detect influenza A-B, RSV, and SARS-CoV-2. There were 115 known positive clinical samples and 35 negative controls obtained from asymptomatic health-care workers included in the study; 25 samples were positive for influenza viruses, 46 for RSV, and 44 for SARS-CoV-2. The sensitivity, specificity, positive predictive value, and negative predictive value of the evaluated method for influenza and SARS-CoV-2 were 100%. The Spearman correlation coefficient was 0.586 (p < 0.05) for influenza and 0.893 (p < 0.05) for SARS-CoV-2. The sensitivity of the aforementioned assay for RSV was 93.47%; the specificity and the positive predictive value were 100%, and the negative predictive value was 92.10%, while the Spearman correlation coefficient was not applicable for the RSV. Overall, the assay under evaluation was shown to be a reliable alternative for the simultaneous detection of influenza viruses, RSV and SARS-CoV-2.

Prospective evaluation of specimen pooling strategy for detection of SARS-CoV-2 using pools of five and six specimens.

The increased demand for SARS-CoV-2 molecular testing during the COVID-19 pandemic resulted in shortage of reagents and consumables. Pooling of specimens could be an alternative strategy to overcome these problems. Initial evaluation of the pooling strategy was performed using known positive specimens, previously tested individually, and their respective pools of plus four (5X), five (6X) and nine (10X) known negative specimens. Subsequently, 35 positive 5X and 35 positive 6X pools containing only one positive specimen per pool were analyzed prospectively regarding the difference in Ct values in pooled versus individual specimens. When the number of samples in the pool were five or six, the average deviation of Ct differences was < 1; therefore, this strategy was followed in the prospective study. Significant difference in Ct values was observed in positive specimens when tested individually and in 5X pools (p = 0.006), while the difference was not significant when positive specimens were tested individually and in 6X pools (p = 0.07). The difference in Ct values was not significant between the 5X and 6X pools. Testing in pools of five or six specimens is a reliable option for SARS-CoV-2 RNA detection when mass testing is needed.

Association Between Upper Respiratory Tract Viral Load, Comorbidities, Disease Severity, and Outcome of Patients With SARS-CoV-2 Infection.

BACKGROUND: There is limited information on the association between upper respiratory tract (URT) viral loads, host factors, and disease severity in SARS-CoV-2-infected patients. METHODS: We studied 1122 patients (mean age, 46 years) diagnosed by polymerase chain reaction (PCR). URT viral load, measured by PCR cycle threshold, was categorized as high, moderate, or low. RESULTS: There were 336 (29.9%) patients with comorbidities; 309 patients (27.5%) had high, 316 (28.2%) moderate, and 497 (44.3%) low viral load. In univariate analyses, compared to patients with moderate or low viral load, patients with high viral load were older, more often had comorbidities, developed Symptomatic disease (COVID-19), were intubated, and died. Patients with high viral load had longer stay in intensive care unit and longer intubation compared to patients with low viral load (P values < .05 for all comparisons). Patients with chronic cardiovascular disease, hypertension, chronic pulmonary disease, immunosuppression, obesity, and chronic neurological disease more often had high viral load (P value < .05 for all comparisons). In multivariate analysis high viral load was associated with COVID-19. Level of viral load was not associated with any other outcome. CONCLUSIONS: URT viral load could be used to identify patients at higher risk for morbidity or severe outcome.

Transmission dynamics of SARS-CoV-2 within families with children in Greece: A study of 23 clusters.

There is limited information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clustering within families with children. We aimed to study the transmission dynamics of SARS-CoV-2 within families with children in Greece. We studied 23 family clusters of coronavirus disease 2019 (COVID-19). Infection was diagnosed by reverse-transcriptase polymerase chain reaction in respiratory specimens. The level of viral load was categorized as high, moderate, or low based on the cycle threshold values. There were 109 household members (66 adults and 43 children). The median attack rate per cluster was 60% (range: 33.4%-100%). An adult member with COVID-19 was the first case in 21 (91.3%) clusters. Transmission of infection occurred from an adult to a child in 19 clusters and/or from an adult to another adult in 12 clusters. There was no evidence of child-to-adult or child-to-child transmission. In total 68 household members (62.4%) tested positive. Children were more likely to have an asymptomatic SARS-CoV-2 infection compared to adults (40% vs 10.5%; P = .021). In contrast, adults were more likely to develop a severe clinical course compared with children (8.8% vs 0%; P = .021). In addition, infected children were significantly more likely to have a low viral load while adults were more likely to have a moderate viral load (40.7% and 18.6% vs 13.8% and 51.7%, respectively; P = .016). In conclusion, while children become infected by SARS-CoV-2, they do not appear to transmit infection to others. Furthermore, children more frequently have an asymptomatic or mild course compared to adults. Further studies are needed to elucidate the role of viral load on these findings.

Bloodstream Infection by Saccharomyces cerevisiae in Two COVID-19 Patients after Receiving Supplementation of Saccharomyces in the ICU.

Co-infections have an unknown impact on the morbidity and mortality of the new clinical syndrome called coronavirus disease 2019 (COVID-19). The syndrome is caused by the new pandemic coronavirus SARS-CoV-2 and it is probably connected with severe traces in the elements of the immune system. Apart from possible Aspergillus infections, particularly in patients with acute respiratory distress syndrome (ARDS), other fungal infections could occur, probably more easily, due to the immunological dysregulation and the critical condition of these patients. Probiotic preparations of Saccharomyces are broadly used for the prevention of antibiotic-associated complications, especially in the intensive care units (ICU). On the other hand, Saccharomyces organisms are reported as agents of invasive infection in immunocompromised or critically ill patients. We report two cases of bloodstream infection by Saccharomyces in two patients hospitalised in the ICU, due to severe COVID-19, after Saccharomyces supplementation.

Influenza vaccine effectiveness against hospitalization with laboratory-confirmed influenza in Greece: A pooled analysis across six seasons, 2013-2014 to 2018-2019.

BACKGROUND: Monitoring seasonal influenza Vaccine Effectiveness (VE) is key to inform vaccination strategies and sustain uptake. Pooling data across multiple seasons increases precision and allows for subgroup analyses, providing more conclusive evidence. Our aim was to assess VE against hospitalization with laboratory-confirmed influenza in Greece over six seasons, from 2013 to 2014 to 2018-2019, using routinely collected surveillance data. METHODS: Swab samples from hospitalized patients across the country were tested for influenza by RT-PCR. We used the test-negative design, with patients testing positive for influenza serving as cases and those testing negative serving as controls. VE was calculated as one minus the Odds Ratio (OR) for influenza vaccination, estimated by mixed-effects logistic regression and adjusted for age, sex, hospitalization type (being in intensive care or not), time from symptom onset to swabbing, and calendar time. Stratified estimates by age and hospitalization type were obtained, and also subgroup estimates by influenza type/subtype and season. Antigenic and genetic characterization of a subset of circulating influenza strains was performed. RESULTS: A total of 3,882 test-positive cases and 5,895 test-negative controls were analyzed. Across all seasons, adjusted VE was 45.5% (95% CI: 31.6-56.6) against all influenza, 62.8% against A(H1N1)pdm09 (95% CI: 40.7-76.7), 28.2% against A(H3N2) (95% CI: 12.0-41.3) and 45.5% against influenza B (95% CI: 29.1-58.1). VE was slightly lower for patients aged 60 years and over, and similar between patients hospitalized inside or outside intensive care. Circulating A(H1N1)pdm09 and B strains were antigenically similar to the vaccine strains, whereas A(H3N2) were not. CONCLUSION: Our results confirm the public health benefits from seasonal influenza vaccination, despite the suboptimal effectiveness against A(H3N2) strains. Continued monitoring of VE is essential, and routinely collected surveillance data can be valuable in this regard.

Circulation of Influenza Type B Lineages in Greece During 2005-2015 and Estimation of Their Impact.

The past decades influenza B lineages Victoria and Yamagata cocirculated. Our aim was to estimate the distribution of the two lineages circulating in Greece and any possible mismatching with vaccine influenza B strains. We studied 490 laboratory-confirmed influenza B nonsevere acute respiratory infection (non-SARI) cases diagnosed in the two National Influenza Reference Laboratories by reverse transcriptase polymerase chain reaction from July 1, 2005 to June 30, 2015 and 100 influenza B SARI cases diagnosed from July 1, 2011 to June 30, 2015. Median matching between the circulating influenza B lineages and the vaccine influenza B strains was 19.2% (range: 0-100%) for non-SARI cases during 2005-2015 and 67.6% (range: 41.2-94.1%) for SARI cases during 2011-2015. In two influenza seasons (2005-2006 and 2006-2007), complete lineage mismatch between influenza B non-SARI cases and influenza B vaccine strains was found. We estimated that 5, 12, or 16 laboratory-confirmed SARI cases could have been prevented by quadrivalent influenza inactivated vaccine (QIV) during the 2011-2012 season and 1, 2, or 3 SARI cases during the 2014-2015 season, with a vaccination coverage rate of 70% and a vaccine effectiveness of 20%, 50%, or 70%, respectively. Significant cocirculation of Victoria and Yamagata B strains and mismatching with vaccine influenza B strains were found during 2005-2015 in Greece. The wide use of a QIV instead of a TIV will confer additional immunity and therefore protection against influenza B, and it is expected to prevent several SARI cases annually. Our findings strongly support the recommendations for using QIV.

Identification of complement-related host genetic risk factors associated with influenza A(H1N1)pdm09 outcome: challenges ahead.

Influenza remains an important threat for human health, despite the extensive study of influenza viruses and the production of effective vaccines. In contrast to virus genetics determinants, host genetic factors with clinical impact remained unexplored until recently. The association between three single nucleotide polymorphisms (SNPs) and influenza outcome in a European population was investigated in the present study. All samples were collected during the influenza A(H1N1)pdm09 post-pandemic period 2010-11 and a sufficient number of severe and fatal cases was included. Host genomic DNA was isolated from pharyngeal samples of 110 patients from northern Greece with severe (n = 59) or mild (n = 51) influenza A(H1N1)pdm09 disease, at baseline, and the genotype of CD55 rs2564978, C1QBP rs3786054 and FCGR2A rs1801274 SNPs was investigated. Our findings suggest a relationship between the two complement-related SNPs, namely, the rare TT genotype of CD55 and the rare AA genotype of C1QBP with increased death risk. No significant differences were observed for FCGR2A genotypes neither with fatality nor disease severity. Additional large-scale genetic association studies are necessary for the identification of reliable host genetic risk factors associated with influenza A(H1N1)pdm09 outcome. Prophylactic intervention of additional high-risk populations, according to their genetic profile, will be a key achievement for the fight against influenza viruses.

Porous dressings of modified chitosan with poly(2-hydroxyethyl acrylate) for topical wound delivery of levofloxacin.

Absorbable and non-absorbable dressings have been fabricated into sponges via a modified thermally induced phase separation method, using a grafted derivative of chitosan with 2-hydroxyethylacrylate (CS-g-PHEA). The material was synthesized via free-radical polymerization and was characterized with FT-IR and (1)H NMR spectroscopies. The swelling ability, biocompatibility and biodegradability of the dressings were evaluated through in vitro assays while antibacterial studies were performed using three different bacterial strains, Methicillin susceptible Staphylococcus aureus (MSSA), Methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Levofloxacin was used as model drug at different concentrations. Morphological characterization of the drug loaded dressings was performed by scanning electron microscopy, while drug-matrix interactions were evaluated by FT-IR spectroscopy. X-ray diffraction studies were carried out for the identification of the physical state for both neat and drug loaded materials. The prepared dressings showed a significant inhibition zone of the bacteria indicating the antibacterial property of the materials and loaded sponges.

Influenza vaccine effectiveness in preventing hospitalizations with laboratory-confirmed influenza in Greece during the 2014-2015 season: A test-negative study.

The 2014-2015 influenza season was marked by circulation of antigenically drifted A/H3N2 strains, raising the possibility of low seasonal influenza Vaccine Effectiveness (VE). We assessed VE against hospitalization with laboratory-confirmed influenza for the 2014-2015 season, using routine surveillance data. Non-sentinel swab samples from Greek hospital inpatients were tested for influenza by RT-PCR in three laboratories, covering the entire country. We estimated VE using a test-negative design. Out of 883 patients with known vaccination status, 161 (18.2%) were vaccinated, and 392/883 patients (44.4%) tested positive for influenza, of whom 162 (41.3%) had type B and 151 (38.5%) had A/H3N2. Adjusted VE was 31.6% (95%CI: 2.9-51.8%) against any influenza, 46.8%, 95%CI: 12.5-67.6%) against type B and -1.9%, 95%CI: -69.5 to 38.7%) against A/H3N2. VE against non-ICU hospitalization appeared to be higher, but the difference did not reach statistical significance. Circulating A/H3N2 viruses showed substantial antigenic drift, while about half of the type B strains were similar to the vaccine strain. Despite the antigenic drift of the A/H3N2 strains, the vaccine still offered substantial protection against hospitalization with laboratory-confirmed influenza, mostly due to a surge in type B influenza late in the season. Vaccine coverage was low, even among groups targeted for vaccination, and considerable effort should be made to improve it. J. Med. Virol. 88:1896-1904, 2016. © 2016 Wiley Periodicals, Inc.

Challenges in antigenic characterization of circulating influenza A(H3N2) viruses during the 2011-2012 influenza season: an ongoing problem?

Genetic and antigenic characterization of 37 representative influenza A(H3N2) virus strains isolated in Greece during the 2011-2012 winter season was performed to evaluate matching of the viruses with the seasonal influenza vaccine strain A/Perth/16/2009. Hemagglutinin gene sequence analysis revealed that all Greek strains clustered within the Victoria/208 genetic clade. Furthermore, substitutions in the antigenic and glycosylation sites suggested potential antigenic drift. Our hemagglutination inhibition (HI) analysis showed that the Greek viruses were Perth/16-like; however, these viruses were characterized as Victoria/208-like when tested at the United Kingdom WHO Collaborating Centre (CC) with HI assays performed in the presence of oseltamivir, a finding consistent with the genetic characterization data. Variability in the HI test performance experienced by other European laboratories indicated that antigenic analysis of the A(H3N2) virus has limitations and, until its standardization, national influenza reference laboratories should include genetic characterization results for selection of representative viruses for detailed antigenic analysis by the WHO CCs.

Influenza vaccine effectiveness against laboratory confirmed influenza in Greece during the 2013-2014 season: a test-negative study.

BACKGROUND: In 2013-2014 Greece experienced a resurgence of severe influenza cases, coincidental with a shift to H1N1pdm09 predominance. We sought to estimate Vaccine Effectiveness (VE) for this season using available surveillance data from hospitals (including both inpatients and outpatients). METHODS: Swab samples were sent by hospital physicians to one of three laboratories, covering the entire country, to be tested for influenza using RT-PCR. The test-negative design was employed, with patients testing positive serving as cases and those testing negative serving as controls. VE was estimated using logistic regression, adjusted for age group, sex, region and calendar time, with further adjustment for unknown vaccination status using inverse response propensity weights. Additional age group stratified estimates and subgroup estimates of VE against H1N1pdm09 and H3N2 were calculated. RESULTS: Out of 1310 patients with known vaccination status, 124 (9.5%) were vaccinated, and 543 patients (41.5%) tested positive for influenza. Adjusted VE was 34.5% (95% CI: 4.1-55.3%) against any influenza, and 56.7% (95% CI: 22.8-75.7%) against H1N1pdm09. VE estimates appeared to be higher for people aged 60 and older, while in those under 60 there was limited evidence of effectiveness. Isolated circulating strains were genetically close to the vaccine strain, with limited evidence of antigenic drift. CONCLUSIONS: These results suggest a moderate protective effect of the 2013-2014 influenza vaccine, mainly against H1N1pdm09 and in people aged 60 and over. Vaccine coverage was very low in Greece, even among groups targeted for vaccination, and substantial efforts should be made to improve it. VE can and should be routinely monitored, and the results taken into account when deciding on influenza vaccine composition for next season.

Genetic analysis of post-pandemic 2010-2011 influenza A(H1N1)pdm09 hemagglutinin virus variants that caused mild, severe, and fatal infections in Northern Greece.

Since its appearance, influenza A(H1N1)pdm09 caused considerable morbidity and mortality in Northern Greece. Genetic analysis of post-pandemic circulating strains scoped to investigate any correlation between genetic variations that emerged during viral evolution and severity of infection. Pharyngeal swabs/aspirates (n = 1,870) were examined with real-time reverse transcription-polymerase chain reaction. Hemagglutinin sequences were analyzed on 110 strains (37 fatal/73 non-fatal cases), followed by statistical and phylogenetic analysis. Influenza A(H1N1)pdm09 was detected in 848 samples. Coexistence of clusters 3, 4, 5, 6, and 7 indicated co-circulation of lineages in Northern Greece. Genetic analysis showed that HA sequences had 96-99% sequence similarity with the vaccine strain and that there was no association between any co-circulating lineage and severity. Several viruses accumulated variations in HA antigenic sites. D222G was significantly associated with fatal infections, supporting its association with increased viral pathogenesis. On the other hand, four variations were associated with milder disease outcomes. Certain signature amino acid changes persisted during and/or after the pandemic, indicating their offer of selective advantages to the virus. Negative selection was observed in 70% of pandemic variations as they probably did not contribute to the virus fitness. It is of interest that persistent variations were highly identified in the vicinity of antigenic or receptor-binding sites. Of those, K171R was associated only with fatal infections. Also of interest, only strains that were isolated from fatal infections had variations that altered both their acid-base and polarity properties. Genetic changes that may alter the antigenicity, pathogenicity and transmissibility of circulating virus variants need to be determined and closely monitored.

Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method.

The study aimed to identify the proportion of tetM-positive Ureaplasma spp. isolates phenotypically susceptible to tetracycline by real-time PCR. Ureaplasma spp. strains of urogenital origin were isolated from 100 female or male adults on A7 agar plates. The presence of Ureaplasma was confirmed by the presence of urease gene by a novel real-time PCR method. Genotyping and sensitivity to tetracyclines were examined using commercial methods. The tetM gene was detected by a novel real-time PCR method especially designed for this study. Ureaplasma parvum was isolated from 87 of the specimens; Ureaplasma urealyticum, from 12; and both species were isolated from a single specimen. All isolates were phenotypically susceptible to tetracyclines. Thirty-five strains were tetM carriers; 29 (82.9%), U. parvum; 5 (14.3%), U. urealyticum; and 1 (2.9%), U. parvum/U. urealyticum. No statistically significant difference was observed between the 3 groups. Four (40%) tetM carriers were isolated from 10 symptomatic men; 11 (32.4%), from 34 symptomatic women; and 20 (35.7%), from 56 asymptomatic women. No statistically significant difference was observed between the 3 groups. The tetM determinant is detected in 35% of phenotypically susceptible to tetracycline Ureaplasma spp. Greek isolates. The use of a real-time PCR technique is particularly helpful, as it makes its detection easy; cost-effective; rapid; and, therefore, more convenient for the surveillance of the dissemination of the tetM resistance gene.

Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections.

The objectives of this research is molecular and phylogenetic analysis of pandemic influenza A(H1N1) 2009 strains that circulated in northern Greece, focusing on severe or fatal infections, identification of sequence variations in relation with the severity of the illness and comparison of circulating viruses with the vaccine strain. A total of 1598 infections were attributed to the novel influenza A(H1N1) virus. Molecular analysis revealed a number of variations at the HA1 sequences of northern Greek circulating strains, some of which were more frequent in viruses that caused severe or fatal infections. Such mutations, the most common being D222G, demand close monitoring to continuously assess associated risks. Phylogenetic analysis confirmed the close match of the majority of circulating strains with A/California/7/09. However it also reveals a trend of 2010 strains to accumulate amino acid variations and form new plylogenetic clades. Constant molecular surveillance is important to monitor the pathogenicity of circulating strains and evaluate the vaccine efficacy.

Absence of human bocavirus in atherosclerotic plaques of carotid arteries by real-time polymerase chain reaction.

An association has been proposed between atherosclerosis and several organisms. We investigated 50 carotid atherosclerotic plaques by real-time polymerase chain reaction for human bocavirus (HBoV). HBoV DNA was not detected in any of the specimens. Future studies are warranted to prove or disprove the role of infectious pathogens, including HBoV, in atherosclerosis.

Molecular and phylogenetic analysis and vaccine strain match of human influenza A(H3N2) viruses isolated in Northern Greece between 2004 and 2008.

Influenza A viruses are characterized by a unique genome structure, causing genetic instability, especially to the genes of haemagglutinin and neuraminidase. The objectives of this research was molecular and phylogenetic analysis of influenza A(H3N2) strains that circulated in Northern Greece since 2004, particularly the identification of sequence variations and the comparison of circulating viruses with vaccine strains. Since 2004 in Northern Greece, a total of 216 clinical samples were positive for influenza virus infections, of which 83 (38.4%) were attributed to influenza A(H3N2). Molecular analysis of the HA genes of 23 isolates showed that all circulating strains had variations at antigenic sites. Receptor binding sites were conserved in 2004-2005 and 2005-2006 strains whereas a variation was observed in all 2006-2007 strains (H195Y). Furthermore, alternative amino acids for sialic acid receptor binding sites were observed in most of the 2004-2006 isolates. Some amino acid substitutions were also observed at the neuraminidase sequences, which however had no effect on the antigenicity of the viruses. Phylogenetic analysis of each year's circulating strains revealed a relatively low match with the vaccine strains A/Fujian/411/02 and A/California/7/04 for 2004-2005 and 2005-2006, respectively, whereas most 2006-2007 isolates match with the vaccine strain, A/Wisconsin/67/05. This year, unique variations were observed at antigenic and glycosylation sites of A/Serres/77/07-like stains. Constant surveillance of yearly variations is of great importance, so that vaccine strains can be evaluated.