Can bronchial secretion cultures identify the etiologic agent of COVID-19-associated pulmonary aspergillosis in ICU patients? Comparison with a species-specific Aspergillus PCR in serum.

Aspergillus spp. isolated from non-BAL cultures of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) patients may reflect colonization rather than infection. Sera (n = 181) from 49 adult ICU CAPA patients (24 probable and 25 possible CAPA) with bronchial secretions (BS) culture positive for Aspergillus spp. were collected and tested for Aspergillus DNA detection by species-specific real-time PCR. Overall, 30/49 (61%) patients were PCR positive. BS culture/serum PCR agreement was moderate (21/30; 70%). Based on serum PCR positive patients, all CAPAs were due to A. fumigatus (80%), A. flavus (10%), and A. terreus (10%). No A. niger/A. nidulans or mixed infections were found despite positive BS cultures.

Discordant results were observed between bronchial secretion cultures and species-specific serum PCR (30%) with A. fumigatus being by far the most common etiological agent of CAPA (80%). No A. niger/A. nidulans or mixed infections were found despite positive cultures.

Variant-Related Differences in Laboratory Biomarkers among Patients Affected with Alpha, Delta and Omicron: A Retrospective Whole Viral Genome Sequencing and Hospital-Setting Cohort Study.

During the COVID-19 pandemic, different SARS-CoV-2 variants of concern (VOC) with specific characteristics have emerged and spread worldwide. At the same time, clinicians routinely evaluate the results of certain blood tests upon patient admission as well as during hospitalization to assess disease severity and the overall patient status. In the present study, we searched for significant cell blood count and biomarker differences among patients affected with the Alpha, Delta and Omicron VOCs at admission. Data from 330 patients were retrieved regarding age, gender, VOC, cell blood count results (WBC, Neut%, Lymph%, Ig%, PLT), common biomarkers (D-dimers, urea, creatinine, SGOT, SGPT, CRP, IL-6, suPAR), ICU admission and death. Statistical analyses were performed using ANOVA, the Kruskal-Wallis test, two-way ANOVA, Chi-square, T-test, the Mann-Whitney test and logistic regression was performed where appropriate using SPSS v.28 and STATA 14. Age and VOC were significantly associated with hospitalization, whereas significant differences among VOC groups were found for WBC, PLT, Neut%, IL-6, creatinine, CRP, D-dimers and suPAR. Our analyses showed that throughout the current pandemic, not only the SARS-CoV-2 VOCs but also the laboratory parameters that are used to evaluate the patient's status at admission are subject to changes.

Antibody response after two doses of the BNT162b2 vaccine among healthcare workers of a Greek Covid 19 referral hospital: A prospective cohort study.

The global vaccination against SARS-CoV-2 has highlighted the need of assessing vaccines' immunogenicity against COVID-19. To evaluate humoral immunity induced by the BNT162b2 vaccine, we enrolled health care workers at AHEPA University Hospital of Thessaloniki, Greece to measure Anti-S SARS-CoV-2, anti-RBD SARS-CoV-2 and neutralizing antibodies. A total of 955 individuals with a median age of 50 years, were included in the study. Median values of antibodies were 1947.27 BAU/mL (Abbott SARS-CoV-2 IgG II Quant), 2064.98 BAU/mL (MAGLUMI SARS-CoV-2 S-RBD IgG) and 2464.63 IU/mL (MAGLUMI SARS-CoV-2 Neutralizing Antibodies). Individuals previously infected had greater antibody responses than infection naive ones and a 7-fold higher neutralizing antibodies titre. Antibodies degreased by age but not sex. Spearman's correlation coefficient among the three assays ranged from 0.903 to 0.969. The BNT162b2 vaccine was highly immunogenic in our cohort. Further research is needed to evaluate the vaccine's immunogenicity through time as well as in different populations.

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.

The Implementation of a Health Care Worker Screening Program Based on the Advanta RT-qPCR Saliva Assay in a Tertiary Care Referral Hospital in Northern Greece.

Health care workers are at increased risk of acquiring SARS-CoV-2 infection due to different exposures in the community and in hospital settings. Interventions implemented to avoid nosocomial outbreaks include preventive testing strategies. In this report, we present results from the mass screening program applied in our hospital to all professionals, irrespective of symptoms or risk of exposure. We processed saliva specimens with real-time reverse transcription polymerase chain reaction. The total number of samples received was 43,726. Positive results were 672 and average positivity rate was 1.21%. The average positivity rate was similar to the positivity rate in the community in Greece and EU. More specifically, 80.5% of the positive participants care for patients in their daily activities, 31% experienced no symptoms before receiving the positive result, 46.1% reported a close contact with a patient or infected coworkers and 32.8% reported a close contact with infected family members. We believe that the identification of asymptomatic carriers has proved the effectiveness of the screening program by preventing the putative nosocomial spread of the virus and the depletion of workforce. In conclusion, in times of high incidence in the community, the periodic testing of health care personnel is wise and relevant for implementation costs.

Dataset from the comparative evaluation of the STANDARD M10 point-of-care analyzer versus the NeuMoDx assay for the molecular diagnosis of SARS-CoV-2.

The STANDARD M10 is a novel cartridge-based real time RT-PCR point of care platform that provides significant advantages regarding SARS-CoV-2 detection including fast turnaround times and no need for specialized personnel and facilities. This assay was recently evaluated in our hospital as a rapid alternative to the already present NeuMoDx assay that is used in everyday practice. For this purpose, 30 nasopharyngeal samples by patients admitted to our hospital were used, regardless of clinical suspicion of COVID-19. In our evaluation, the sensitivity of STANDARD M10 was 95%, the specificity was 100%, the positive predictive value was 100%, the negative predictive value was 90% and the kappa coefficient of agreement was 0.927 (p < 0.001).

Negative Predictive Value of the Rapid Test Ag 2019-nCoV During the Predominance of Omicron over the Delta Variant and Implications in the Emergency Department

The high prevalence of asymptomatic patients infected with SARS-CoV-2 during the pandemic peaks and the common occurrence of in-hospital transmission urges the need for SARS-CoV-2 testing before admission of all patients with non-COVID-related symptoms. RT-PCR testing however is costly, time-consuming, and increases the length of stay in the emergency department. For the aforementioned reasons, we propose that the admission of non-suspected COVID-19 patients to the appropriate department should be based on the sole use of the rapid test result. In order to assess the safety of this suggestion, we assessed the negative predictive value of our rapid antigen tests that was calculated at 96.38%. This value was considered acceptable and the proposed strategy was applied in our hospital improving the overall turnaround times. However, since various rapid tests may perform differently, we propose that hospitals assess their own methodologies before implementing our proposal.

Microbiological characteristics of bacteremias among COVID-19 hospitalized patients in a tertiary referral hospital in Northern Greece during the second epidemic wave.

Northern Greece was struck by an intense second COVID-19 (coronavirus disease 2019) epidemic wave during the fall of 2020. Because of the coinciding silent epidemic of multidrug-resistant organisms, the handling of COVID-19 patients became even more challenging. In the present study, the microbiological characteristics of bacteremias in confirmed cases of hospitalized COVID-19 patients were determined. Data from 1165 patients hospitalized between September and December 2020 were reviewed regarding the frequency of bloodstream infections, the epidemiology and the antibiotic susceptibility profiles of the causative bacteria. The hospital's antibiotic susceptibility data for all major nosocomial pathogens isolated from bacteremias of COVID-19 patients between September and December 2020 versus those between September and December 2019 were also compared. Overall, 122 patients developed bacteremia (10.47%). The average of time interval between hospitalization date and development of bacteremia was 13.98 days. Admission to ICU occurred in 98 out of 122 patients with an average stay time of 15.85 days and 90.81% in-hospital mortality. In total, 166 pathogens were recovered including 114 Gram-negative bacteria and 52 Gram-positive cocci. Acinetobacter baumannii was the most frequent (n = 51) followed by Klebsiella pneumoniae (n = 45) and Enterococcus faecium (n = 31). Bacteremias in hospitalized COVID-19 patients were related with prolonged time of hospitalization and higher in-hospital mortality, and the isolated microorganisms represented the bacterial species that were present in our hospital before the COVID-19 pandemic. Worryingly, the antibiotic resistance rates were increased compared with the pre-pandemic era for all major opportunistic bacterial pathogens. The pandemic highlighted the need for continuous surveillance of patients with prolonged hospitalization.

Molecular and Clinical Prognostic Biomarkers of COVID-19 Severity and Persistence.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), poses several challenges to clinicians, due to its unpredictable clinical course. The identification of laboratory biomarkers, specific cellular, and molecular mediators of immune response could contribute to the prognosis and management of COVID-19 patients. Of utmost importance is also the detection of differentially expressed genes, which can serve as transcriptomic signatures, providing information valuable to stratify patients into groups, based on the severity of the disease. The role of biomarkers such as IL-6, procalcitonin, neutrophil-lymphocyte ratio, white blood cell counts, etc. has already been highlighted in recently published studies; however, there is a notable amount of new evidence that has not been summarized yet, especially regarding transcriptomic signatures. Hence, in this review, we assess the latest cellular and molecular data and determine the significance of abnormalities in potential biomarkers for COVID-19 severity and persistence. Furthermore, we applied Gene Ontology (GO) enrichment analysis using the genes reported as differentially expressed in the literature in order to investigate which biological pathways are significantly enriched. The analysis revealed a number of processes, such as inflammatory response, and monocyte and neutrophil chemotaxis, which occur as part of the complex immune response to SARS-CoV-2.

Early initiation of the respiratory syncytial virus season in 2021-2022, Greece.

Respiratory syncytial virus (RSV) is the most common viral pathogen causing respiratory disease in the pediatric population. An unexpected sudden upsurge of RSV infections among children was observed in September 2021 in Greece. Forty-one rhinopharyngeal samples from children under the age of 2 years with confirmed RSV bronchiolitis were tested to identify the genotype(s) of the RSV strain(s). The children were hospitalized during September-November 2021 in three tertiary hospitals in northern Greece. A one-step RT-PCR which amplifies a fragment of the second hypervariable region of the G protein gene was applied. PCR products were sequenced, and phylogenetic analysis was performed. Most (80.5%) RSV cases were typed as RSV-A, with RSV-B accounting for 19.5% of cases. RSV-A and RSV-B sequences clustered within the ON1 and BA genotypes, respectively. As the same genotypes were detected in cases observed during 2016-2018 in northern Greece, it was suggested that the early upsurge of infections was not related to the emergence of novel strain(s), but it was the result of the absence of immunity among children and their mothers due to the restriction measures taken during the COVID-19 pandemic in the previous RSV season. Awareness is needed to diagnose even the out-of-season RSV infections, while molecular epidemiology plays a key role in monitoring the efficacy of currently available therapeutics and for those under development.

Microbiological characteristics of bacteremias among COVID-19 hospitalized patients in a tertiary referral hospital in Northern Greece during the second epidemic wave

Northern Greece was struck by an intense second COVID-19 epidemic wave during the fall of 2020. Because of the coinciding silent epidemic of MDR organisms the handling of COVID-19 patients became even more challenging. In the present study, the microbiological characteristics of bacteraemias in confirmed cases of hospitalized COVID-19 patients were determined. Data from 1165 patients hospitalized between September and December 2020 were reviewed regarding the frequency of bloodstream infections, the epidemiology and the antibiotic susceptibility profiles of the causative bacteria. The hospital's antibiotic susceptibility data for all major nosocomial pathogens isolated from bacteremias of COVID-19 patients between September-December 2020 versus September-December 2019 bacteremias were also compared. Overall, 122 patients developed bacteremia (10.47%). The average of time interval between hospitalization date and development of bacteremia was 13.98 days. Admission to ICU occurred in 98 out of 122 patients with an average time of stay 15.85 days and 90.81% in-hospital mortality. In total, 166 pathogens were recovered including 114 Gram-negative bacteria and 52 Gram-positive cocci. Acinetobacter baumannii were the most frequent (n = 51) followed by Klebsiella pneumoniae (n = 45) and Enterococcus faecium (n = 31). Bacteremias in hospitalized COVID-19 patients were related with prolonged time of hospitalization and higher in-hospital mortality and the isolated microorganisms represented the bacterial species that were present in our hospital before the COVID-19 pandemic. Worryingly, the antibiotic resistance rates were increased compared to the pre-pandemic era for all major opportunistic bacterial pathogens. The pandemic highlighted the need for continuous surveillance of patients with prolonged hospitalization.

Evaluation of the QuantiFERON SARS-CoV-2 assay to assess cellular immunogenicity of the BNT162b2 mRNA COVID-19 vaccine in individuals with low and high humoral response.

Vaccines against SARS-CoV-2 are known to be less immunogenic for some individuals, whereas others present notably high levels of antibody production. We assessed the cellular response to BNT162b2 among individuals with low post-vaccination antibody levels as well as in a small group of individuals with high titers. Antibody levels were assessed by the Abbott SARS-CoV-2 IgG II Quant assay. The interferon-γ production of T-cells in response to SARS-CoV-2 antigens was determined using Qiagen's QuantiFERON SARS-CoV-2 ELISA test. Our results showed that participants with high antibody levels presented adequate cellular response in all studied cases, whereas those with low antibody levels generally showed limited to almost absent cellular response five months post vaccination.

Evaluation of the Advanta Dx SARS-CoV-2 RT-PCR Assay, a High-Throughput Extraction-Free Diagnostic Test for the Detection of SARS-CoV-2 in Saliva: A Diagnostic Accuracy Study.

Nasopharyngeal swab specimen (NPS) molecular testing is considered the gold standard for SARS-CoV-2 detection. However, saliva is an attractive, noninvasive specimen alternative. The aim of the study was to evaluate the diagnostic accuracy of Advanta Dx SARS-CoV-2 RT-PCR saliva-based assay against paired NPS tested with either NeumoDxTM SARS-CoV-2 assay or Abbott Real Time SARS-CoV-2 assay as the reference method. We prospectively evaluated the method in two settings: a diagnostic outpatient and a healthcare worker screening convenience sample, collected in November-December 2020. SARS-CoV-2 was detected in 27.7% (61/220) of diagnostic samples and in 5% (10/200) of screening samples. Overall, saliva test in diagnostic samples had a sensitivity of 88.5% (77.8-95.3%) and specificity of 98.1% (94.6-99.6%); in screening samples, the sensitivity was 90% (55.5-99.7%) and specificity 100% (98.1-100%). Our data suggests that the Fluidigm Advanta Dx RT-PCR saliva-based assay may be a reliable diagnostic tool for COVID-19 diagnosis in symptomatic individuals and screening asymptomatic healthcare workers.

A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain.

Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log10 copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.

COVID-19 and Antimicrobial Resistance: Data from the Greek Electronic System for the Surveillance of Antimicrobial Resistance-WHONET-Greece (January 2018-March 2021).

Changes in hospitals' daily practice due to COVID-19 pandemic may have an impact on antimicrobial resistance (AMR). We aimed to assess this possible impact as captured by the Greek Electronic System for the Surveillance of Antimicrobial Resistance (WHONET-Greece). Routine susceptibility data of 17,837 Gram-negative and Gram-positive bacterial isolates from blood and respiratory specimens of hospitalized patients in nine COVID-19 tertiary hospitals were used in order to identify potential differences in AMR trends in the last three years, divided into two periods, January 2018-March 2020 and April 2020-March 2021. Interrupted time-series analysis was used to evaluate differences in the trends of non-susceptibility before and after the changes due to COVID-19. We found significant differences in the slope of non-susceptibility trends of Acinetobacter baumannii blood and respiratory isolates to amikacin, tigecycline and colistin; of Klebsiella pneumoniae blood and respiratory isolates to meropenem and tigecycline; and of Pseudomonas aeruginosa respiratory isolates to imipenem, meropenem and levofloxacin. Additionally, we found significant differences in the slope of non-susceptibility trends of Staphylococcus aureus isolates to oxacillin and of Enterococcus faecium isolates to glycopeptides. Assessing in this early stage, through surveillance of routine laboratory data, the way a new global threat like COVID-19 could affect an already ongoing pandemic like AMR provides useful information for prompt action.

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions.

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.