Athanasios Kossyvakis

A case of imported Middle East Respiratory Syndrome coronavirus infection and public health response, Greece, April 2014

On 18 April 2014, a case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection was laboratory confirmed in Athens, Greece in a patient returning from Jeddah, Saudi Arabia. Main symptoms upon initial presentation were protracted fever and diarrhoea, during hospitalisation he developed bilateral pneumonia and his condition worsened. During 14 days prior to onset of illness, he had extensive contact with the healthcare environment in Jeddah. Contact tracing revealed 73 contacts, no secondary cases had occurred by 22 April.

Guidance for clinical and public health laboratories testing for influenza virus antiviral drug susceptibility in Europe

Two classes of antiviral drugs are licensed in Europe for treatment and prophylaxis of influenza; the M2 ion-channel blockers amantadine and rimantadine acting against type A influenza viruses only and the neuraminidase enzyme inhibitors zanamivir and oseltamivir acting against type A and type B influenza viruses. This guidance document was developed for but not limited to the European Union (EU) and other European Economic Area (EEA) countries on how and when to test for influenza virus antiviral drug susceptibility. It is aimed at clinical and influenza surveillance laboratories carrying out antiviral drug susceptibility testing on influenza viruses from patients suspected of harbouring viruses with reduced susceptibility or for the monitoring of the emergence of such among circulating viruses, respectively. Therefore, the guidance should not be read as a directive or an algorithm for treatment. Monitoring for emergence of influenza viruses with reduced drug susceptibility in hospitalized cases is crucial for decision making on possible changes to antiviral treatment. Therefore, it is important to test for antiviral susceptibility in certain patient groups, such as patients treated with influenza antiviral drugs. It is also important to determine the frequency of viruses with natural (not related to drug use) reduced susceptibility among community and hospitalized cases, as this knowledge is essential for making empirical antiviral treatment decisions. Furthermore, testing of specimens from community influenza patients is needed to determine the frequency of viruses with reduced susceptibility and good viral fitness that are readily transmissible, as they may become dominant among circulating viruses. Phenotypic neuraminidase enzyme inhibition assays are recommended to determine the level of inhibition of the neuraminidase enzyme by antiviral drugs as a measure of drug susceptibility of the virus. Genotypic assays are recommended to identify amino acid substitutions in the neuraminidase and M2 ion-channel proteins that have been associated with reduced antiviral susceptibility previously. By 2012 all circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses were naturally resistant to the M2 ion-channel blockers, so priority should be given to testing for neuraminidase inhibitor susceptibility.

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.

Challenges in Antigenic Characterization of Circulating Influenza A(H3N2) Viruses during the 2011-2012 Influenza Season: an Ongoing Problem?

ABSTRACT 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 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.

Laboratory investigation and phylogenetic analysis of an imported Middle East respiratory syndrome coronavirus case in Greece.

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.

Genetic variability of human metapneumo- and bocaviruses in children with respiratory tract infections

The genotypic analysis of human metapneumo‐(HMPV) and boca‐(HBoV) viruses circulating in Greece and their comparison to reference and other clinical strains.

NAIplot: An opensource web tool to visualize neuraminidase inhibitor (NAI) phenotypic susceptibility results using kernel density plots

The results of neuraminidase inhibitor (NAI) enzyme inhibition assays are commonly expressed as 50% inhibitory concentration (IC50) fold-change values and presented graphically in box plots (box-and-whisker plots). An alternative and more informative type of graph is the kernel density plot, which we propose should be the preferred one for this purpose. In this paper we discuss the limitations of box plots and the advantages of the kernel density plot, and we present NAIplot, an opensource web application that allows convenient creation of density plots specifically for visualizing the results of NAI enzyme inhibition assays, as well as for general purposes.

Improving influenza virological surveillance in Europe: strain-based reporting of antigenic and genetic characterisation data, 11 European countries, influenza season 2013/14

Influenza antigenic and genetic characterisation data are crucial for influenza vaccine composition decision making. Previously, aggregate data were reported to the European Centre for Disease Prevention and Control by European Union/European Economic Area (EU/EEA) countries. A system for collecting case-specific influenza antigenic and genetic characterisation data was established for the 2013/14 influenza season. In a pilot study, 11 EU/EEA countries reported through the new mechanism. We demonstrated feasibility of reporting strain-based antigenic and genetic data and ca 10% of influenza virus-positive specimens were selected for further characterisation. Proportions of characterised virus (sub)types were similar to influenza virus circulation levels. The main genetic clades were represented by A/StPetersburg/27/2011(H1N1)pdm09 and A/Texas/50/2012(H3N2). A(H1N1)pdm09 viruses were more prevalent in age groups (by years) < 1 (65%; p = 0.0111), 20–39 (50%; p = 0.0046) and 40–64 (55%; p = 0.00001) while A(H3N2) viruses were most prevalent in those ≥ 65 years (62%*; p = 0.0012). Hospitalised patients in the age groups 6–19 years (67%; p = 0.0494) and ≥ 65 years (52%; p = 0.0005) were more frequently infected by A/Texas/50/2012 A(H3N2)-like viruses compared with hospitalised cases in other age groups. Strain-based reporting enabled deeper understanding of influenza virus circulation among hospitalised patients and substantially improved the reporting of virus characterisation data. Therefore, strain-based reporting of readily available data is recommended to all reporting countries within the EU/EEA.

Mixed viral infections of the respiratory tract; an epidemiological study during consecutive winter seasons

The current study aimed to describe the molecular epidemiology of mixed respiratory viral infections during consecutive winter seasons in a tertiary care hospital. Patients with symptoms of respiratory tract infection were evaluated during the 2009‐2011 and 2013‐15 winter seasons. A clinical microarray technique was used for viral detection. Clinical and epidemiological data were correlated with mixed viral detection and the need for hospitalization. In 332 out of 604 (54.4%) evaluated patients (17.6% children) a respiratory virus was identified. Mixed viral infections were diagnosed in 68/332 (20.5%) patients with virus detection (66.2% mixed Influenza‐RSV infections). Mixed viral infections were more commonly detected in children (OR 3.7; 95%CI 1.9‐5.6, P < 0.01) and patients with comorbidities. In logistic regression analyses, mixed viral infections were associated with younger age (mean age 30.4 years vs. 41.8 years, P ≤ 0.001) and increased rates of fever (OR: 2.7; 95%CI 1.04‐7.2, P < 0.05) but no adverse outcomes or increased rates of hospitalization. High rates of mixed viral infections were noted during all winter seasons (especially Influenza and RSV) and were more common in younger patients. The clinical significance of mixed respiratory viral infection needs further elucidation.