Seasonal and inter-seasonal RSV activity in the European Region during the COVID-19 pandemic from autumn 2020 to summer 2022.

Background: The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in early 2020 and subsequent implementation of public health and social measures (PHSM) disrupted the epidemiology of respiratory viruses. This work describes the epidemiology of respiratory syncytial virus (RSV) observed during two winter seasons (weeks 40-20) and inter-seasonal periods (weeks 21-39) during the pandemic between October 2020 and September 2022. Methods: Using data submitted to The European Surveillance System (TESSy) by countries or territories in the World Health Organization (WHO) European Region between weeks 40/2020 and 39/2022, we aggregated country-specific weekly RSV counts of sentinel, non-sentinel and Severe Acute Respiratory Infection (SARI) surveillance specimens and calculated percentage positivity. Results for both 2020/21 and 2021/22 seasons and inter-seasons were compared with pre-pandemic 2016/17 to 2019/20 seasons and inter-seasons. Results: Although more specimens were tested than in pre-COVID-19 pandemic seasons, very few RSV detections were reported during the 2020/21 season in all surveillance systems. During the 2021 inter-season, a gradual increase in detections was observed in all systems. In 2021/22, all systems saw early peaks of RSV infection, and during the 2022 inter-seasonal period, patterns of detections were closer to those seen before the COVID-19 pandemic. Conclusion: RSV surveillance continued throughout the COVID-19 pandemic, with an initial reduction in transmission, followed by very high and out-of-season RSV circulation (summer 2021) and then an early start of the 2021/22 season. As of the 2022/23 season, RSV circulation had not yet normalised.

Can variants, reinfection, symptoms and test types affect COVID-19 diagnostic performance? A large-scale retrospective study of AG-RDTs during circulation of Delta and Omicron variants, Czechia, December 2021 to February 2022.

BackgroundThe sensitivity and specificity of selected antigen detection rapid diagnostic tests (AG-RDTs) for SARS-CoV-2 were determined in the unvaccinated population when the Delta variant was circulating. Viral loads, dynamics, symptoms and tissue tropism differ between Omicron and Delta.AimWe aimed to compare AG-RDT sensitivity and specificity in selected subgroups during Omicron vs Delta circulation.MethodsWe retrospectively paired AG-RDT results with PCRs registered in Czechia's Information System for Infectious Diseases from 1 to 25 December 2021 (Delta, n = 20,121) and 20 January to 24 February 2022 (Omicron, n = 47,104).ResultsWhen confirmatory PCR was conducted on the same day as AG-RDT as a proxy for antigen testing close to peak viral load, the average sensitivity for Delta was 80.4% and for Omicron 81.4% (p < 0.05). Sensitivity in vaccinated individuals was lower for Omicron (OR = 0.94; 95% confidence interval (CI): 0.87-1.03), particularly in reinfections (OR = 0.83; 95% CI: 0.75-0.92). Saliva AG-RDT sensitivity was below average for both Delta (74.4%) and Omicron (78.4%). Tests on the European Union Category A list had higher sensitivity than tests in Category B. The highest sensitivity for Omicron (88.5%) was recorded for patients with loss of smell or taste, however, these symptoms were almost 10-fold less common than for Delta. The sensitivity of AG-RDTs performed on initially asymptomatic individuals done 1, 2 or 3 days before a positive PCR test was consistently lower for Omicron compared with Delta.ConclusionSensitivity for Omicron was lower in subgroups that may become more common if SARS-CoV-2 becomes an endemic virus.

Role of population and test characteristics in antigen-based SARS-CoV-2 diagnosis, Czechia, August to November 2021.

BackgroundAnalyses of diagnostic performance of SARS-CoV-2 antigen rapid diagnostic tests (AG-RDTs) based on long-term data, population subgroups and many AG-RDT types are scarce.AimWe aimed to analyse sensitivity and specificity of AG-RDTs for subgroups based on age, incidence, sample type, reason for test, symptoms, vaccination status and the AG-RDT's presence on approved lists.MethodsWe included AG-RDT results registered in Czechia's Information System for Infectious Diseases between August and November 2021. Subpopulations were analysed based on 346,000 test results for which a confirmatory PCR test was recorded ≤ 3 days after the AG-RDT; 38 AG-RDTs with more than 100 PCR-positive and 300 PCR-negative samples were individually evaluated.ResultsAverage sensitivity and specificity were 72.4% and 96.7%, respectively. We recorded lower sensitivity for age groups 0-12 (65.5%) and 13-18 years (65.3%). The sensitivity level rose with increasing SARS-CoV-2 incidence from 66.0% to 76.7%. Nasopharyngeal samples had the highest sensitivity and saliva the lowest. Sensitivity for preventive reasons was 63.6% vs 86.1% when testing for suspected infection. Sensitivity was 84.8% when one or more symptoms were reported compared with 57.1% for no symptoms. Vaccination was associated with a 4.2% higher sensitivity. Significantly higher sensitivity levels pertained to AG-RDTs on the World Health Organization Emergency Use List (WHO EUL), European Union Common List and the list of the United Kingdom's Department of Health and Social Care.ConclusionAG-RDTs from approved lists should be considered, especially in situations associated with lower viral load. Results are limited to SARS-CoV-2 delta variant.

Retrospective Analysis Revealed an April Occurrence of Monkeypox in the Czech Republic.

Herein, we present our findings of an early appearance of the Monkeypox virus in Prague, Czech Republic. A retrospective analysis of biological samples, carried out on the 28th of April, revealed a previously unrecognized case of Monkeypox virus (MPxV) infection. Subsequent data analysis confirmed that the virus strain belongs to the ongoing outbreak. Combined with clinical and epidemiological investigations, we extended the roots of the current outbreak at least back to 16th of April, 2022.

Monkeypox and Herpes Simplex Virus Type 2 Coinfection: Case Report of Perianal Lesions in HIV-Positive Patient.

ABSTRACT: We report a case of monkeypox and herpes simplex type 2 coinfection in an HIV-positive male patient who has sex with men. This case report describes a diagnostic approach for papular rash in the anal area of the male patient who has sex with men with a history of sexually transmitted disease. This is also the first documented case of monkeypox in the Czech Republic, which was confirmed after a retrospective review of swab samples from a previously hospitalized HIV-positive patient.

Reverse-zoonotic transmission of SARS-CoV-2 lineage alpha (B.1.1.7) to great apes and exotic felids in a zoo in the Czech Republic.

We report an outbreak of SARS-CoV-2 lineage alpha in gorillas and felid species in a zoo in Prague, Czech Republic. The course of illness and clinical signs are described, as are the results of characterization of these particular SARS-CoV-2 variants by next-generation sequencing and phylogenetic analysis. The putative transmission routes are also discussed.

Multi-branch cooperation on SARS-CoV-2 omicron capture: a case report.

Shortly after the WHOs first notice a suspected case of omicron SARS-CoV-2 was reported in Liberec, Czech Republic. The primary goal of the following actions was to test the presence of the variant and stop the spread of the virus variant. On November 25 a sixty-year-old lady, who had recently returned from Namibia, visited a GP with flu-like symptoms and a rash on her chest. The antigen test was positive for SARS-CoV-2, a PCR test was planned. At that time, it was not known that a new variant of concern was spreading from Africa. On November 26 in the morning the GP announced a suspected omicron case to the Regional public health authority, who organized the following steps. A mobile sampling team was sent to the patient's home immediately, sample transported into the regional hospital and analyzed with the help of the national reference laboratory. The captured virus SARS-CoV-2 fitted the description of the omicron variant, was shared in the GISAID database and named hCoV-19/Czech Republic/KNL_2021-110119140/2021. Contact tracing was started immediately, eleven persons were tested and quarantined. One of them positive with no further spread. It is the first documented omicron case in the Czech Republic and one of the first cases in Europe, with an excellent systemic response to the alert. The laboratory was able to detect the omicron variant instantly after the request. This case also demonstrates how easily the virus spreads on long distances and how important it might be to increase the uptake of the booster vaccine.

A universal RT-qPCR assay for "One Health" detection of influenza A viruses.

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses.

The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.

Circulation of influenza A and B in the Czech Republic from 2000-2001 to 2015-2016.

BACKGROUND: To improve national influenza vaccination recommendations, additional data on influenza A and B virus circulation are needed. Here, we describe the circulation of influenza A and B in the Czech Republic during 16 seasons. METHODS: This was a retrospective analysis of data collected from the 2000-2001 to 2015-2016 influenza seasons by the Czech Republic national influenza surveillance network. Influenza was confirmed and viral isolates subtyped by virological assays followed by antigen detection or by reverse transcriptase-polymerase chain reaction. RESULTS: Of 16,940 samples collected, 5144 (30.4%) were influenza-positive. Influenza A represented 78.6% of positive cases overall and accounted for more than 55.0% of all influenza cases in every season, except for 2005-2006 (6.0%). Both A/H1N1 and A/H3N2 were detected in most seasons, except for 2001-2002 and 2003-2004 (only A/H3N2), and 2007-2008 and 2009-2010 (only A/H1N1). Influenza B represented 21.4% of positive cases overall (range, 0.0-94.0% per season). Both influenza B lineages were detected in three seasons, a single B lineage in 11, and no B strain in two. For the 11 seasons where influenza B accounted for ≥20% of positive cases, the dominant lineage was Yamagata in six and Victoria in four. In the remaining season, the two lineages co-circulated. For two seasons (2005-2006 and 2007-2008), the B lineage in the trivalent influenza vaccine did not match the dominant circulating B lineage. CONCLUSIONS: In the Czech Republic, during the 2000-2001 to 2015-2016 influenza seasons, influenza virus circulation varied considerably. Although influenza A accounted for the most cases in almost all seasons, influenza B made a substantial, sometimes dominant, contribution to influenza disease.

Correction: MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

[This corrects the article DOI: 10.1371/journal.pone.0151204.].

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay.

Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Mumps in the Czech Republic in 2013: Clinical Characteristics, Mumps Virus Genotyping, and Epidemiological Links.

AIM: The aim of the study was to map the incidence of mumps in the Czech Republic in terms of clinical symptoms, epidemiological links, and characteristics of circulating genotypes. METHODS: Patients with suspected mumps examined in the Infectious Diseases Clinic of the Na Bulovce Hospital in 2013 were enrolled in the study. Buccal swab specimens were tested by means of nucleic acid detection (RT-qPCR) and when positive, they were cultured in tissue culture. Sequencing was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit and Genetic Analyzer 3500. The SeqScape software was used for the analysis of sequencing data and filtering out low quality reads. The phylogenetic analysis and genotyping were performed using the Mega 6 software. To generate the phylogenetic tree, all sequences were aligned by the MAFFT tool and the alignment obtained was edited using the BioEdit software. In all patients, selected biochemical markers (C-reactive protein, white blood cell count and serum amylase) were measured. The EPIDAT system used for reporting infectious diseases, record keeping, and data analysis in the Czech Republic was the source of statistical data. RESULTS: Eighty-nine patients with suspected mumps were examined in the Na Bulovce Hospital and 65 of them were laboratory confirmed with mumps: 40 males (61.5%) and 25 females (38.5%). The mean age of the study cohort was 25.9 years (median age of 23 years, age range from 10 to 73 years) and 14 patients were under 18 years of age. Thirty-four (52.3%) patients were vaccinated in childhood, 28 (43.1%) were unvaccinated, and for three persons, vaccination data were not available. A severe course of the disease was reported in 15 (23.1%) patients. Fourteen of them needed hospitalization because of orchitis (9 males) and meningitis (5 patients). One patient with orchitis was treated on an outpatient basis. The need for hospitalization tended to be lower in the unvaccinated patients (14.7% vs. 35.7%, p=0.076). In 2013, 1,553 cases of mumps were reported to the EPIDAT system. Of these, 640 were laboratory confirmed. The most often reported complications were orchitis (90 cases, i.e. 10.3%) and meningitis (21 cases, i.e. 1.4%). Orchitis was diagnosed in 30.3% of the unvaccinated and in 6.4% of the vaccinated males. Meningitis occurred in 3.1% of the unvaccinated and in 1.0% of the vaccinated patients. CONCLUSION: Despite the emergence of mumps among the vaccinated population, the present study has confirmed a positive effect of the vaccine, particularly on the incidence of complications and inflammatory markers. All 30 sequenced mumps virus strains were assigned to group G. A secondary vaccine failure due to waning immunity seems to be a plausible explanation for the rise in mumps cases.

Large-scale nucleotide sequence alignment and sequence variability assessment to identify the evolutionarily highly conserved regions for universal screening PCR assay design: an example of influenza A virus.

The development of a diagnostic polymerase chain reaction (PCR) or quantitative PCR (qPCR) assay for universal detection of highly variable viral genomes is always a difficult task. The purpose of this chapter is to provide a guideline on how to align, process, and evaluate a huge set of homologous nucleotide sequences in order to reveal the evolutionarily most conserved positions suitable for universal qPCR primer and hybridization probe design. Attention is paid to the quantification and clear graphical visualization of the sequence variability at each position of the alignment. In addition, specific problems related to the processing of the extremely large sequence pool are highlighted. All of these steps are performed using an ordinary desktop computer without the need for extensive mathematical or computational skills.

Local-scale diversity and between-year "frozen evolution" of avian influenza A viruses in nature.

Influenza A virus (IAV) in wild bird reservoir hosts is characterized by the perpetuation in a plethora of subtype and genotype constellations. Multiyear monitoring studies carried out during the last two decades worldwide have provided a large body of knowledge regarding the ecology of IAV in wild birds. Nevertheless, other issues of avian IAV evolution have not been fully elucidated, such as the complexity and dynamics of genetic interactions between the co-circulating IAV genomes taking place at a local-scale level or the phenomenon of frozen evolution. We investigated the IAV diversity in a mallard population residing in a single pond in the Czech Republic. Despite the relative small number of samples collected, remarkable heterogeneity was revealed with four different IAV subtype combinations, H6N2, H6N9, H11N2, and H11N9, and six genomic constellations in co-circulation. Moreover, the H6, H11, and N2 segments belonged to two distinguishable sub-lineages. A reconstruction of the pattern of genetic reassortment revealed direct parent-progeny relationships between the H6N2, H11N9 and H6N9 viruses. Interestingly the IAV, with the H6N9 subtype, was re-detected a year later in a genetically unchanged form in the close proximity of the original sampling locality. The almost absolute nucleotide sequence identity of all the respective genomic segments between the two H6N9 viruses indicates frozen evolution as a result of prolonged conservation in the environment. The persistence of the H6N9 IAV in various abiotic and biotic environmental components was also discussed.

[Phylogenetic analysis and genotyping of A/H3N2 Influenza viruses isolated from patients hospitalised with influenza-like illness symptoms in the na bulovce hospital in the season 2011/2012]. / Fylogenetická analýza a genotypizace viru chripky A/H3N2 izolovaných od pacientu hospitalizovaných s príznaky ILI (Influenza-Like Illnesses) v Nemocnici Na Bulovce v sezone 2011/2012.

Influenza A virus is an important cause of acute respiratory infections (ARI). Clinical manifestations of ARI vary from mild or moderate to life-threatening conditions requiring intensive care. Given the segmented genome, a large natural reservoir of other influenza virus subtypes, and antibody selection pressure in the population, the virus is variable and genetically unstable. The phylogenetic analysis and genotyping of A/H3N2 influenza viruses isolated from patients hospitalised with influenza-like illness symptoms in the Na Bulovce Hospital in the season 2011/2012 support the assumption that the pathogenicity is a polygenic trait modifiable by the host health status and seems not to be unambiguously associated with any specific mutations.

[Microneutralization assay in the diagnosis of influenza infection]. / Mikroneutralizace v diagnostice chripkové infekce.

Serology plays an important role in the diagnosis of influenza, particularly in the detection of post-vaccination and post-infection antibodies. When considering the range of diagnostic options, the serological method should be selected depending on the circumstances - whether single or paired serum samples are tested, whether adequate patient medical history data are available, whether epidemiological links are suspected, and, in particular, to what purpose the result will be used (differential diagnosis, post-infection follow-up, post-vaccination monitoring, etc.). The virus neutralization assay is one of the most sensitive and most objective serological tests, but it is highly dependent on the reaction balance and quality of the virus used. Determining the protective titer is crucial for the routine practice. Based on our experiments, we concluded that the virus neutralizing antibody titers are up to eight times as high in comparison with the hemaglutination inhibition test (HIT) or complement fixation reaction (CFR), but the correlation varies and is significantly influenced by interindividual variation in anti-neuraminidase antibodies and those against some internal proteins of influenza virus. We assume that the protective titer in the virus neutralization assay will be not less than 1:80. The predictive value of the titers below 1:40 is questionable.