Publications by authors named "Anna Papa"

204 Publications

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples.

J Clin Virol 2021 Aug 8;141:104908. Epub 2021 Jul 8.

Clinical Microbiological Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands. Electronic address:

Introduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories.

Methods: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed.

Results: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection.

Conclusion: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.
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http://dx.doi.org/10.1016/j.jcv.2021.104908DOI Listing
August 2021

Genetic characterization of two methicillin-resistant Staphylococcus aureus spa type t127 strains isolated from workers in the dairy production chain in Greece.

Acta Microbiol Immunol Hung 2021 Jun 15. Epub 2021 Jun 15.

1Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Greece.

Methicillin-resistant Staphylococcus aureus (MRSA) constitutes a constant threat for the public health. Aim of the present study was to analyse the whole genome sequences of two MRSA strains belonging to Staphylococcus protein A (spa) type t127 isolated from humans working in two distantly located dairy production farms in Greece.MRSA strains were isolated from the nasal cavity of a food handler in a milk industry in Epirus, northwestern Greece (E-MRSA), and a person working in a cattle farm in Thrace, northeastern Greece (T-MRSA). Whole genome sequences taken using next generation sequencing were analysed for resistance and virulence genes applying various bioinformatic tools.Both isolates were assigned to ST1-IVa-t127 type, and they were transferring genes conferring resistance to tetracycline, β-lactams, and aminoglycosides; T-MRSA was carrying additional genes leading to macrolide, lincosamide and streptogramin B (MLSB) resistance. Both isolates were carrying three plasmid replicon types, rep5, rep7 and rep16, while T-MRSA harboured also rep10 and rep15. E-MRSA carried scn and sak genes which were absent from T-MRSA.In conclusion, the genetic characterization of two unrelated ST1-IVa-t127 MRSA strains isolated from humans in close contact with livestock in Greece can be used as basis for further epidemiological and evolutionary studies.
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http://dx.doi.org/10.1556/030.2021.01460DOI Listing
June 2021

West Nile fever upsurge in a Greek regional unit, 2020.

Acta Trop 2021 Sep 12;221:106010. Epub 2021 Jun 12.

Ecodevelopment SA, Thessaloniki, Greece.

During the 2020 West Nile virus (WNV) transmission season, Greece was the most affected EU Member State. More than one third of human cases occurred in Serres regional unit in northern Greece, which is characterized by the presence of a major wetland (Kerkini lake and Strimon river). A total of 2809 Culex pipiens mosquitoes collected in Serres were grouped into 70 pools and tested for WNV. Ten (14.3%) pools were found positive, and all WNV sequences belonged to the Central European subclade of WNV lineage 2. The first human case occurred in a village nearby the lake, and all following cases occurred across the connected river and its tributaries. Similar distribution presented the sites where WNV-positive mosquitoes were detected. The number of Culex spp. mosquitoes per trap per night was higher in 2020 than in previous years (2017-2019). The spatial and temporal distribution of human cases and WNV-positive mosquitoes in 2020 in Serres regional unit suggest that the upsurge of the virus circulation was probably related with factors that affected the ecosystem of the wetland.
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http://dx.doi.org/10.1016/j.actatropica.2021.106010DOI Listing
September 2021

Recommendations for the introduction of metagenomic next-generation sequencing in clinical virology, part II: bioinformatic analysis and reporting.

J Clin Virol 2021 May 26;138:104812. Epub 2021 Mar 26.

Virology Laboratory, Genomics and Health Area, Centre for Public Health Research (FISABIO-Public Health), Valencia, Spain; Department of Microbiology, Medical School, University of Valencia, Spain; CIBERESP, Instituto de Salud Carlos III, Madrid, Spain. Electronic address:

Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS is still in its early stages of broader translation into clinical applications. To further support the development, implementation, optimization and standardization of mNGS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mNGS for viral diagnostics to share methodologies and experiences, and to develop application guidelines. Following the ENNGS publication Recommendations for the introduction of mNGS in clinical virology, part I: wet lab procedure in this journal, the current manuscript aims to provide practical recommendations for the bioinformatic analysis of mNGS data and reporting of results to clinicians.
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http://dx.doi.org/10.1016/j.jcv.2021.104812DOI Listing
May 2021

Spread of NDM-producing Klebsiella pneumoniae in a tertiary Greek hospital.

Acta Microbiol Immunol Hung 2021 Mar 3. Epub 2021 Mar 3.

2Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece.

Bacterial carbapenem resistance, especially when mediated by transferable carbapenemases, is of important public health concern. An increased number of metallo-β-lactamase (MBL)-producing Klebsiella pneumoniae strains isolated in a tertiary hospital in Thessaloniki, Greece, called for further genetic investigation.The study included 29 non-repetitive carbapenem resistant K. pneumoniae isolates phenotypically characterized as MBL-producers collected in a tertiary hospital in Greece. The isolates were screened for the detection of carbapenemase genes (K. pneumoniae carbapenemase (blaKPC), Verona-integron-encoded MBL-1 (blaVIM-1), imipenemase (blaIMP), oxacillinase-48 (blaOXA-48) and New Delhi MBL (blaNDM)). The genetic relationship of the isolates was determined by Random Amplified Polymorphic DNA (RAPD) analysis. The whole genome sequences (WGS) from two NDM-positive K. pneumoniae isolates were further characterized.The presence of New Delhi MBL (blaNDM) gene was confirmed in all K. pneumoniae isolates, while blaKPC and blaVIM-1 genes were co-detected in one and two isolates, respectively. The RAPD analysis showed that the isolates were clustered into two groups. The whole genome sequence analysis of two K. pneumoniae isolates revealed that they belonged to the sequence type 11, they carried the blaNDM-1 gene, and exhibited differences in the number and type of the plasmids and the resistant genes.All MBL-producing K. pneumoniae isolates of the study harbored a blaNDM gene, while WGS analysis revealed genetic diversity in resistance genes. Continuous surveillance is needed to detect the emergence of new clones in a hospital setting, while application of antimicrobial stewardship is the only way to reduce the spread of multi-resistant bacteria.
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http://dx.doi.org/10.1556/030.2021.01400DOI Listing
March 2021

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

J Infect Dis 2021 04;223(7):1132-1138

Department of Microbiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.

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.
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http://dx.doi.org/10.1093/infdis/jiaa804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798974PMC
April 2021

Children and Adolescents With SARS-CoV-2 Infection: Epidemiology, Clinical Course and Viral Loads.

Pediatr Infect Dis J 2020 12;39(12):e388-e392

Second Department of Pediatrics, National and Kapodistrian University of Athens, Aglaia Kyriakou Children's Hospital, Athens, Greece.

Background: There is limited information on severe acute respiratory syndrome virus 2 (SARS-CoV-2) infection in children.

Methods: We retrieved data from the national database on SARS-CoV-2 infections. We studied in-family transmission. The level of viral load was categorized as high, moderate, or low based on the cycle threshold values.

Results: We studied 203 SARS-CoV-2-infected children (median age: 11 years; range: 6 days to 18.4 years); 111 (54.7%) had an asymptomatic infection. Among the 92 children (45.3%) with coronavirus disease 2019 (COVID-19), 24 (26.1%) were hospitalized. Infants <1 year were more likely to develop COVID-19 (19.5% of all COVID-19 cases) (P-value = 0.001). There was no significant difference between viral load and age, sex, underlying condition, fever and hospitalization, as well as between type of SARS-CoV-2 infection and age, sex, underlying condition and viral load. Transmission from a household member accounted for 132 of 178 (74.2%) children for whom the source of infection was identified. An adult member with COVID-19 was the first case in 125 (66.8%) family clusters. Child-to-adult transmission was found in one occasion only.

Conclusions: SARS-CoV-2 infection is mainly asymptomatic or mild during childhood. Adults appear to play a key role in spread of the virus in families. Most children have moderate or high viral loads regardless of age, symptoms or severity of infection. Further studies are needed to elucidate the role of children in the ongoing pandemic and particularly in light of schools reopening and the need to prioritize groups for vaccination, when COVID-19 vaccines will be available.
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http://dx.doi.org/10.1097/INF.0000000000002899DOI Listing
December 2020

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

Arch Virol 2020 Dec 4;165(12):3023-3072. Epub 2020 Sep 4.

Colorado State University, Fort Collins, CO, USA.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.
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http://dx.doi.org/10.1007/s00705-020-04731-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606449PMC
December 2020

ICTV Virus Taxonomy Profile: .

J Gen Virol 2020 08 24;101(8):798-799. Epub 2020 Aug 24.

USAMRIID, Fort Detrick, Frederick, Maryland, USA.

Members of the family produce enveloped virions with three single-stranded RNA segments comprising 17.1 to 22.8 kb in total. These viruses are maintained in arthropods and transmitted by ticks to mammals or birds. Crimean-Congo hemorrhagic fever virus is tick-borne and is endemic in most of Asia, Africa, Southern and Eastern Europe whereas Nairobi sheep disease virus, which is also tick-borne, causes lethal haemorrhagic gastroenteritis in small ruminants in Africa and India. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family , which is available at ictv.global/report/nairoviridae.
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http://dx.doi.org/10.1099/jgv.0.001485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641396PMC
August 2020

West Nile virus in humans, Greece, 2018: the largest seasonal number of cases, 9 years after its emergence in the country.

Euro Surveill 2020 08;25(32)

National Reference Center for Arboviruses and Haemorrhagic Fever Viruses, Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece.

BackgroundHuman cases of West Nile virus (WNV) infection are recorded since 2010 in Greece, with seasonal outbreaks occurring almost annually. Enhanced surveillance has been implemented since 2010, to promptly characterise cases' temporal and geographical distribution and inform authorities for implementation of appropriate measures (mosquito control, health education, blood safety).AimWe describe the epidemiology of WNV human infections in Greece focusing on the 2018 season.MethodsThe National Public Health Organization advised physicians to test all suspect WNV infection cases and refer samples to reference laboratories. Laboratories notified diagnosed cases on a daily basis. Treating physicians, patients, and infected blood donors were interviewed within 48 hours after diagnosis and the probable infection location was identified. Hospitalised cases were followed up until discharge.ResultsA total of 317 autochthonous WNV infection cases were diagnosed in 2018. Among them, 243 cases had neuroinvasive disease (WNND), representing a 23% increase of WNND cases compared with 2010, the previous most intense season. There were 51 deaths. Cases started occurring from week 22, earlier than usual. Both rural and urban areas were affected, with 86 (26% of the total) municipalities belonging to seven (54% of the total) regions recording cases. Two major epicentres were identified in Attica and Central Macedonia regions.ConclusionsThe largest number of human cases of WNV infection ever recorded in Greece occurred in 2018, with a wide geographical distribution, suggesting intense virus circulation. Enhanced surveillance is vital for the early detection of human cases and the prompt implementation of response measures.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.32.1900543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427301PMC
August 2020

Isolation and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) from milk of dairy goats under low-input farm management in Greece.

Vet Microbiol 2020 Aug 20;247:108749. Epub 2020 Jun 20.

Laboratory of Food Hygiene-Veterinary Public Health, School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece. Electronic address:

The presence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in raw milk is a challenge for veterinarians and public health professionals. In this study, we investigated the presence and clonality of S. aureus and MRSA in milk of individual dairy goats with subclinical mastitis reared under the low-input farming system in Greece and determined the isolates' enterotoxin gene carriage and their ability to form biofilms. S. aureus was isolated from 162 out of the 559 milk samples examined (29 %) and one isolate per S. aureus-positive sample was further characterized. S. aureus isolates were very closely related even among farms of distant geographical regions. Nine S. aureus isolates carried a functional mecA gene and were classified as MRSA. The S. aureus protein A (spa) typing in the MRSA isolates showed that four belonged to spa type t127 (44.4 %), three to t2049 (33.3 %) and two to t7947 (22.2 %). The spa type t7947 is reported for the first time in Greece. The MRSA isolates originated from two very distantly located farms, one located in the island of Skopelos and the other in Central Macedonia. Four of the MRSA isolates carried the staphylococcal enterotoxin genes sea or sec. Most of the isolates (92 % of S. aureus and 77.8 % of the MRSA) possessed moderate or weak biofilm-formation ability. Raw milk from low-input goat herds may serve as a potential vector of antimicrobial-resistant S. aureus to raw-milk consumers.
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http://dx.doi.org/10.1016/j.vetmic.2020.108749DOI Listing
August 2020

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

J Med Virol 2021 03 26;93(3):1414-1420. Epub 2020 Aug 26.

Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece.

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.
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http://dx.doi.org/10.1002/jmv.26394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441283PMC
March 2021

Application of 16S rRNA next generation sequencing in ticks in Greece.

Heliyon 2020 Jul 28;6(7):e04542. Epub 2020 Jul 28.

Clinic of Farm Animals, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Greece.

Tick-borne bacteria pose a significant threat to human and veterinary public health. Greece is a Mediterranean country with rich tick fauna and the most commonly detected tick-borne bacterial pathogens are members of the and species. The variable V2-V4 and V6-V9 regions of 16S rRNA gene of seven ticks belonging to four genera representative in Greece () were analysed using multiple primer pairs by next generation sequencing (NGS). Nine bacterial phyla corresponding to 95 families, 116 genera and 172 species were identified. Proteobacteria was the predominant phylum in five of the seven ticks, followed by Actinobacteria, which predominated in two ticks. The tick-borne bacteria included and species, while " Midichloria mitochondrii" were detected in high abundance in ticks and less in ; -like endosymbionts were detected in , , and less in ticks. Co-infections with and were also observed. 16S rRNA NGS is a powerful tool to investigate the tick bacteriome and can improve the strategies for prevention and control of tick-borne diseases.
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http://dx.doi.org/10.1016/j.heliyon.2020.e04542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393430PMC
July 2020

Human visceral leishmaniasis in northern Greece: Seroepidemiology and risk factors in endemic region.

J Vector Borne Dis 2019 Jul-Sep;56(3):244-251

Department of Microbiology, Medical School of Aristotle University of Thessaloniki, Thessaloniki, Greece.

Background & Objectives: Visceral leishmaniasis is endemic in Greece, with sporadic cases reported annually both in the mainland and in coastal areas. Seroepidemiological studies across Greece report seropositivity rates from 0.5 to 15%, in different parts of the country. The aim of the present study was to evaluate the Leishmania seropositivity rate of the general population of Drama prefecture, a rich in water supply region, in northern Greece.

Methods: Serum samples collected from 347 healthy individuals were tested for IgG Leishmania infantum antibodies. Furthermore, 132 domestic dogs, clinically suspected to suffer from canine leishmaniasis, from all across the prefecture, were also evaluated.

Results: Among 347 healthy individuals tested, 24 (6.9%) were positive for IgG L. infantum antibodies. Age, gender, occupational and leisure time activities didn't show significant relation to IgG seropositivity, whereas low altitude of place of residency and residency at places with surface water were significantly related to IgG seropositivity. All seropositive individuals follow a geographic pattern, gathering themselves in Drama basin (rich in surface and underground water bodies), whereas canine leishmaniasis cases show a wide distribution across the prefecture.

Interpretation & Conclusion: Evaluation of both human seroprevalence and high incidence of canine leishmaniasis, as well as favorable landscape and climatic conditions of the study area, indicates that high level of clinical awareness need to be employed by physicians, as human and canine visceral leishmaniasis constitutes a serious public health concern.
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http://dx.doi.org/10.4103/0972-9062.289399DOI Listing
August 2020

Delayed Laboratory Response to COVID-19 Caused by Molecular Diagnostic Contamination.

Emerg Infect Dis 2020 08 20;26(8):1944-1946. Epub 2020 May 20.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.
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http://dx.doi.org/10.3201/eid2608.201843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392437PMC
August 2020

West Nile virus lineage 2 in Culex mosquitoes in Thessaly, Greece, 2019.

Acta Trop 2020 May 15;208:105514. Epub 2020 May 15.

EcoDevelopment SA, Thessaloniki, Greece.

West Nile virus is a flavivirus transmitted to humans mainly by mosquito bites. Outbreaks are observed in several European countries, and Greece is one of the most affected countries during the recent years. Thessaly was one of the most affected regions in Greece in 2019. A total of 3,025 Culex spp. mosquitoes collected in Thessaly were grouped into 47 pools and tested for West Nile virus (WNV). Eight (17%) pools were found positive. Whole genome sequences were obtained from two positive pools. Phylogenetic analysis showed that the causative strain was an evolutionary variant of the strains circulating in 2018 belonging to the Balkan subgroup of the Central European subclade of WNV lineage 2.
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http://dx.doi.org/10.1016/j.actatropica.2020.105514DOI Listing
May 2020

Respiratory Syncytial Virus in Greece, 2016-2018.

Intervirology 2019 29;62(5-6):210-215. Epub 2020 Apr 29.

Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece,

Human respiratory syncytial virus (RSV) is the leading cause of acute bronchiolitis in infants and young children. Children under the age of 2 years, hospitalized for bronchiolitis in the pediatric clinic of a tertiary hospital in northern Greece, were tested for RSV infection during two RSV seasons (2016-2017 and 2017-2018). RSV was detected in 37 of 71 (52.1%) patients, most of them younger than 6 months. Both RSV subtypes were detected - RSV-A (54.1%) and RSV-B (45.9%) - with predominance of RSV-A during the 2016-2017 and RSV-B during the 2017-2018 season. RSV-A and RSV-B sequences clustered within the ON1 and BA genotypes, respectively. Compared to the prototype strains, several amino acid substitutions were observed in the duplication region of the G gene. The study provides a first insight into the molecular epidemiology of RSV in Greece.
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http://dx.doi.org/10.1159/000506049DOI Listing
April 2020

West Nile virus lineage 2 in humans and mosquitoes in Bulgaria, 2018-2019.

J Clin Virol 2020 06 11;127:104365. Epub 2020 Apr 11.

Department of Parasitology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria.

Background: West Nile virus (WNV) lineage 2, and especially the Hungarian clade, predominates in Europe. Most of the Hungarian clade strains cluster into 2 groups: Central/South-West European and Balkan.

Objectives: Since there was not any study on WNV in mosquitoes in Bulgaria, the present study was designed to test Culex spp. mosquitoes in areas near the Danube river. The aim of the study was to gain an insight into the recent molecular epidemiology of WNV in Bulgaria.

Study Design: A total of 1871 Culex pipiens mosquitoes collected in 2018 and clinical samples from 23 patients with West Nile neuroinavsive disease observed in 2018 and 2019 were tested by TaqMan RT-PCR and RT-nested PCR and PCR products were sequenced.

Results: WNV RNA was detected in clinical samples from 10 patients and in five (12.2 %) of 41 pools of Cx. pipiens mosquitos by realtime RT-PCR, resulting in a minimum infection rate of mosquitoes of 0.27 %. Phylogenetic analysis based on partial NS3 gene sequences from one clinical sample and four mosquito pools showed that all sequences clustered into the Hungarian clade of WNV lineage 2 and all but one were identical to respective sequences from Romania. Whole genome sequences of one mosquito pool belong to the Hungarian group of WNV lineage 2 and cluster in a separate subclade from the Bulgarian strain from 2015, suggesting that at least two different introductions occurred in Bulgaria.

Conclusions: The current study provides insights into the geographic distribution of WNV in Bulgaria.
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http://dx.doi.org/10.1016/j.jcv.2020.104365DOI Listing
June 2020

Specialist laboratory networks as preparedness and response tool - the Emerging Viral Diseases-Expert Laboratory Network and the Chikungunya outbreak, Thailand, 2019.

Euro Surveill 2020 04;25(13)

Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.13.1900438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140599PMC
April 2020

West Nile neuroinvasive disease. Report of four cases in Northern Greece, 2018.

J Med Virol 2020 08 11;92(8):1322-1325. Epub 2020 Mar 11.

1st Department of Neurology, AHEPA Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece.

West Nile virus (WNV) is a mosquito-borne RNA flavivirus which caused several epidemics worldwide. The year 2018 was a WNV record year for Europe, including Greece, with earlier and longer transmission season with higher than the previous number of cases. It has been proposed that some simple biochemical markers may be helpful for the recognition of WNV neuroinvasive disease, its differential from other neurological infectious diseases and prognosis. We describe four cases that suffered from WNV meningitis and/or encephalitis hospitalized in 2018 in a tertiary hospital in Thessaloniki, Greece, and investigate the importance of simple biomarkers for the recognition of WNV etiology.
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http://dx.doi.org/10.1002/jmv.25739DOI Listing
August 2020

Meeting report: Eleventh International Conference on Hantaviruses.

Antiviral Res 2020 04 15;176:104733. Epub 2020 Feb 15.

KU Leuven, Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, National Reference Center for Hantaviruses, Leuven, Belgium.

The 2019 11th International Conference on Hantaviruses (ICH 2019) was organized by the International Society for Hantaviruses (ISH), and held on September 1-4, 2019, at the Irish College, in Leuven, Belgium. These ICHs have been held every three years since 1989. ICH 2019 was attended by 158 participants from 33 countries. The current report summarizes research presented on all aspects of hantavirology: ecology; pathogenesis and immune responses; virus phylogeny, replication and morphogenesis; epidemiology; vaccines, therapeutics and prevention; and clinical aspects and diagnosis.
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http://dx.doi.org/10.1016/j.antiviral.2020.104733DOI Listing
April 2020

Laboratory readiness and response for novel coronavirus (2019-nCoV) in expert laboratories in 30 EU/EEA countries, January 2020.

Euro Surveill 2020 02 11;25(6). Epub 2020 Feb 11.

The participating members of EVD-LabNet and ERLI-Net are acknowledged at the end of the article.

Timely detection of novel coronavirus (2019-nCoV) infection cases is crucial to interrupt the spread of this virus. We assessed the required expertise and capacity for molecular detection of 2019-nCoV in specialised laboratories in 30 European Union/European Economic Area (EU/EEA) countries. Thirty-eight laboratories in 24 EU/EEA countries had diagnostic tests available by 29 January 2020. A coverage of all EU/EEA countries was expected by mid-February. Availability of primers/probes, positive controls and personnel were main implementation barriers.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.6.2000082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029448PMC
February 2020

Toscana, West Nile, Usutu and tick-borne encephalitis viruses: external quality assessment for molecular detection of emerging neurotropic viruses in Europe, 2017.

Euro Surveill 2019 12;24(50)

Unite des Virus Emergents (UVE: Aix Marseille Univ, IRD 190, INSERM 1207, IHU Mediterranee Infection), Marseille, France.

BackgroundNeurotropic arboviruses are increasingly recognised as causative agents of neurological disease in Europe but underdiagnosis is still suspected. Capability for accurate diagnosis is a prerequisite for adequate clinical and public health response.AimTo improve diagnostic capability in EVD-LabNet laboratories, we organised an external quality assessment (EQA) focusing on molecular detection of Toscana (TOSV), Usutu (USUV), West Nile (WNV) and tick-borne encephalitis viruses (TBEV).MethodsSixty-nine laboratories were invited. The EQA panel included two WNV RNA-positive samples (lineages 1 and 2), two TOSV RNA-positive samples (lineages A and B), one TBEV RNA-positive sample (Western subtype), one USUV RNA-positive sample and four negative samples. The EQA focused on overall capability rather than sensitivity of the used techniques. Only detection of one, clinically relevant, concentration per virus species and lineage was assessed.ResultsThe final EQA analysis included 51 laboratories from 35 countries; 44 of these laboratories were from 28 of 31 countries in the European Union/European Economic Area (EU/EEA). USUV diagnostic capability was lowest (28 laboratories in 18 countries), WNV detection capacity was highest (48 laboratories in 32 countries). Twenty-five laboratories were able to test the whole EQA panel, of which only 11 provided completely correct results. The highest scores were observed for WNV and TOSV (92%), followed by TBEV (86%) and USUV (75%).ConclusionWe observed wide variety in extraction methods and RT-PCR tests, showing a profound absence of standardisation across European laboratories. Overall, the results were not satisfactory; capacity and capability need to be improved in 40 laboratories.
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.50.1900051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918591PMC
December 2019

Detection of flaviviruses and alphaviruses in mosquitoes in Central Macedonia, Greece, 2018.

Acta Trop 2020 Feb 20;202:105278. Epub 2019 Nov 20.

EcoDevelopment SA, Thessaloniki, Greece.

Culex mosquitoes are vectors of several flaviviruses and alphaviruses posing a potential risk to public and veterinary health. In order to gain an insight into the flaviviruses and alphaviruses circulating in the five regional units of Central Macedonia in northern Greece, 17,470 female Culex spp. mosquitoes collected during 2018 were tested for these viruses. Among 229 mosquito pools, West Nile virus (WNV) was detected in 10 (4.4%) pools, while insect-specific flavi- and alphaviruses were detected in 2 (0.9%) and 8 (3.5%) pools, respectively. WNV minimum infection rate (MIR) was 0.57. The highest MIR was identified in Thessaloniki regional unit, where several human cases of WNV infection occurred in 2018. All ten WNV sequences cluster into the Central European subclade of lineage 2. It is of note that the first WNV-positive mosquito pool was detected two weeks prior the report of the first human case in the area, suggesting that testing of mosquitoes could serve as early warning system.
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http://dx.doi.org/10.1016/j.actatropica.2019.105278DOI Listing
February 2020

Epstein-Barr Encephalitis in a Child with Congenital Human Immunodeficiency Virus Infection: A Case Report Calling for No Forgetfulness.

Curr HIV Res 2020 ;18(1):63-66

3rd Department of Pediatrics, Faculty of Medicine, Aristotle University School of Health Sciences, Hippokration General Hospital of Thessaloniki, Thessaloniki, Greece.

Background: In resource-rich settings, the rate of mother-to-child transmission of human immunodeficiency virus (HIV) has dramatically decreased by virtue of a combination of preventive strategies during the last two decades.

Case Presentation: We present a case of progressive developmental milestone loss in a toddler with previously unknown congenitally acquired human immunodeficiency virus (HIV) infection, complicated by an Epstein-Barr virus (EBV) coinfection.

Conclusion: Our report underscores the differential diagnosis between HIV encephalopathy and EBV encephalitis and the vertical transmission of the HIV infection, which constitutes an alarming issue in terms of public health.
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http://dx.doi.org/10.2174/1570162X17666191017101223DOI Listing
June 2021

Geographical Variability Affects CCHFV Detection by RT-PCR: A Tool for In-Silico Evaluation of Molecular Assays.

Viruses 2019 10 16;11(10). Epub 2019 Oct 16.

National Institute for Infectious Diseases (INMI) "L. Spallanzani" IRCCS, WHO Collaborating Center for clinical care, diagnosis, response and training on Highly Infectious Diseases, 00149 Rome, Italy.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient's travel history and geographic distribution of the different CCHFV strains.
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http://dx.doi.org/10.3390/v11100953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833031PMC
October 2019

Evolutionary dynamics of lineage 2 West Nile virus in Europe, 2004-2018: Phylogeny, selection pressure and phylogeography.

Mol Phylogenet Evol 2019 12 12;141:106617. Epub 2019 Sep 12.

Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece. Electronic address:

West Nile virus (WNV) is an arbovirus causing neuroinvasive disease to humans and equines. Since 2004, lineage 2 WNV strains have been identified in Europe and have been implicated in severe outbreaks, with that of 2018 exceeding the total number from the previous seven years. The aim of this study was to explore the evolutionary process that shapes the genetic diversity of lineage 2 WNV strains (belonging to the Central European/Hungarian subclade) and reconstruct the origin and transmission routes in Europe, and especially in the Balkans. For this purpose, a high number of whole genome sequences (WGSs) were analyzed, along with newly characterized sequences, including strains from the 2018 WNV transmission season in Greece. Maximum likelihood and Bayesian inference methods were used to perform the phylogenetic and phylodynamic analyses and phylogeographic reconstruction. The majority of the Central European/Hungarian lineage 2 strains are grouped in 2 phylogenetic subgroups (Central/South-West European and Balkan) with bush-like topology. Purifying selection shapes their evolution, however, strong evidence of positive selection was revealed in 7 non-structural protein codons of NS1, NS4B and NS5. Thirty-two amino-acid substitutions were fixed in different phylogenetic subgroups, indicating that random genetic drift is responsible for the majority of evolutionary changes. Virus migration, followed by subsequent local evolution is responsible for continuously evolving strains throughout Europe. In total, 10 virus transitions between discrete geographical locations, involving virus spread from Central Europe to other regions, were highly supported. Three novel, independent introductions from Hungary and Bulgaria were responsible for the 2018 re-emergence of WNV in Northern Greece, indicating that Hungary remains an important ecological niche for the virus and has a central role for the dissemination of novel strains in the Balkans. In Northern Greece, tMRCA estimations indicated that a 1-to 2-year period of silent enzootic transmission precedes spread to dead-end hosts. Reconstruction of WNV population dynamics, from WGS data, revealed epidemic patterns characterized by 3- to 5-year oscillations in Europe. Future studies are necessary to determine the possible driving factors for these fluctuations i.e. avian herd immunity and climatic conditions affecting mosquito and bird populations. Maintaining adequate epidemiological surveillance with emphasis on obtaining WGS data, in areas at risk, is crucial for understanding the epidemiology and transmission patterns of WNV. It can further support integrated programs for risk assessment of virus circulation dynamics, aiming to targeted prevention and response measures for veterinary and public health in Europe.
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http://dx.doi.org/10.1016/j.ympev.2019.106617DOI Listing
December 2019

A case of Crimean-Congo haemorrhagic fever imported in Greece: Contact tracing and management of exposed healthcare workers.

J Infect Prev 2019 Jul 6;20(4):171-178. Epub 2019 Jun 6.

Department of Infection Control, University Hospital of Alexandroupolis, Alexandroupolis, Greece.

Background: Nosocomial transmission is a major mode of infection of Crimean-Congo haemorrhagic fever (CCHF). In May 2018, a patient with CCHF was hospitalised in Greece.

Objective: Our aim was to present the management of healthcare workers (HCWs) to the CCHF case.

Methods: Contact tracing, risk assessment and follow-up of exposed HCWs were performed. Testing (RT-PCR and/or serology) was offered to contacts. Post-exposure prophylaxis (PEP) with ribavirin was considered for high-risk exposures.

Results: Ninety-one HCWs were exposed to the case. Sixty-six HCWs were grouped as high-risk exposures. Ribavirin PEP was offered to 29 HCWs; seven agreed to receive prophylaxis. Forty-one HCWs were tested for CCHF infection; none was found positive. Gaps in infection control occurred.

Discussion: CCHF should be considered in patients with compatible travel history and clinical and laboratory findings. Early clinical suspicion and laboratory confirmation are imperative for the implementation of appropriate infection control measures. Ribavirin should be considered for high-risk exposures. Infection control capacity for highly pathogenic agents should increase.
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http://dx.doi.org/10.1177/1757177419852666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683607PMC
July 2019
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