Mild Hyperglycaemia in Hospitalised Children with Moderate COVID-19 Infection.

Background and Objectives: COVID-19 infection may influence many physiological processes, including glucose metabolism. Acute hyperglycaemia has been related to a worse prognosis in patients with severe COVID-19 infection. The aim of our study was to find out if moderate COVID-19 infection is associated with hyperglycaemia. Materials and Methods: A total of 235 children were enrolled in the study between October 2021 and October 2022, 112 with confirmed COVID-19 infection and 123 with other RNA viral infection. In all patients, types of symptoms, glycaemia at the time of admission, and basic anthropometric and biochemical parameters were recorded. Results: Average glycaemia was significantly higher in COVID-19 patients compared to other viral infections (5.7 ± 1.12 vs. 5.31 ± 1.4 mmol/L, p = 0.011). This difference was more obvious in subgroups with gastrointestinal manifestations (5.6 ± 1.11 vs. 4.81 ± 1.38 mmol/L, p = 0.0006) and with fever (5.76±1.22 vs. 5.11±1.37 mmol/L, p = 0.002), while no significant difference was found in subgroups with mainly respiratory symptoms. The risk of hyperglycaemia (>5.6 mmol/L) was higher in COVID-19 patients compared to other viral infections (OR = 1.86, 95%CI = 1.10-3.14, p = 0.02). The risk of hyperglycaemia was significantly higher in COVID-19 compared to other viral infections in the subgroups of patients with fever (OR = 3.59, 95% CI 1.755-7.345, p = 0.0005) and with gastrointestinal manifestations (OR = 2.48, 95% CI 1.058-5.791, p = 0.036). Conclusion: According to our results, mild hyperglycaemia was significantly more common in children with moderate COVID-19 infection compared to other RNA virus respiratory and gastrointestinal infections, especially when accompanied by fever or gastrointestinal symptoms.

Comparison of the methods for isolation and detection of SARS-CoV-2 RNA in municipal wastewater.

Introduction: Coronavirus SARS-CoV-2 is a causative agent responsible for the current global pandemic situation known as COVID-19. Clinical manifestations of COVID-19 include a wide range of symptoms from mild (i.e., cough, fever, dyspnea) to severe pneumonia-like respiratory symptoms. SARS-CoV-2 has been demonstrated to be detectable in the stool of COVID-19 patients. Waste-based epidemiology (WBE) has been shown as a promising approach for early detection and monitoring of SARS-CoV-2 in the local population performed via collection, isolation, and detection of viral pathogens from environmental sources. Methods: In order to select the optimal protocol for monitoring the COVID-19 epidemiological situation in region Turiec, Slovakia, we (1) compared methods for SARS-CoV-2 separation and isolation, including virus precipitation by polyethylene glycol (PEG), virus purification via ultrafiltration (Vivaspin®) and subsequent isolation by NucleoSpin RNA Virus kit (Macherey-Nagel), and direct isolation from wastewater (Zymo Environ Water RNA Kit); (2) evaluated the impact of water freezing on SARS- CoV-2 separation, isolation, and detection; (3) evaluated the role of wastewater filtration on virus stability; and (4) determined appropriate methods including reverse transcription-droplet digital PCR (RT-ddPCR) and real-time quantitative polymerase chain reaction (RT-qPCR) (targeting the same genes, i.e., RdRp and gene E) for quantitative detection of SARS-CoV-2 in wastewater samples. Results: (1) Usage of Zymo Environ Water RNA Kit provided superior quality of isolated RNA in comparison with both ultracentrifugation and PEG precipitation. (2) Freezing of wastewater samples significantly reduces the RNA yield. (3) Filtering is counterproductive when Zymo Environ Water RNA Kit is used. (4) According to the specificity and sensitivity, the RT-ddPCR outperforms RT-qPCR. Discussion: The results of our study suggest that WBE is a valuable early warning alert and represents a non-invasive approach to monitor viral pathogens, thus protects public health on a regional and national level. In addition, we have shown that the sensitivity of testing the samples with a nearer detection limit can be improved by selecting the appropriate combination of enrichment, isolation, and detection methods.

Sensitive SARS-CoV-2 detection, air travel Covid-19 testing, variant determination and fast direct PCR detection, using ddPCR and RT-qPCR methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monitoring in air traffic is important in the prevention of the virus spreading from abroad. The gold standard for SARS-CoV-2 detection is RT-qPCR; however, for early and low viral load detection, a much more sensitive method, such as droplet digital PCR (ddPCR), is required. Our first step was to developed both, ddPCR and RT-qPCR methods, for sensitive SARS-CoV-2 detection. Analysis of ten swab/saliva samples of five Covid-19 patients in different stages of disease showed positivity in 6/10 samples with RT-qPCR and 9/10 with ddPCR. We also used our RT-qPCR method for SARS-CoV-2 detection without the need of RNA extraction, obtaining results in 90-120 minutes. We analyzed 116 self-collected saliva samples from passengers and airport staff arriving from abroad. All samples were negative by RT-qPCR, while 1 was positive, using ddPCR. Lastly, we developed ddPCR assays for SARS-CoV-2 variants identification (alpha, beta, gamma, delta/kappa) that are more economically advantageous when compared to NGS. Our findings demonstrated that saliva samples can be stored at ambient temperature, as we did not observe any significant difference between a fresh sample and the same sample after 24 hours (p = 0.23), hence, saliva collection is the optimal route for sampling airplane passengers. Our results also showed that droplet digital PCR is a more suitable method for detecting virus from saliva, compared to RT-qPCR. Keywords: COVID-19; RT-PCR; ddPCR; SARS-CoV-2; nasopharyngeal swab; saliva.

Systematic Genomic Surveillance of SARS-CoV-2 Virus on Illumina Sequencing Platforms in the Slovak Republic-One Year Experience.

To explore a genomic pool of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the pandemic, the Ministry of Health of the Slovak Republic formed a genomics surveillance workgroup, and the Public Health Authority of the Slovak Republic launched a systematic national epidemiological surveillance using whole-genome sequencing (WGS). Six out of seven genomic centers implementing Illumina sequencing technology were involved in the national SARS-CoV-2 virus sequencing program. Here we analyze a total of 33,024 SARS-CoV-2 isolates collected from the Slovak population from 1 March 2021, to 31 March 2022, that were sequenced and analyzed in a consistent manner. Overall, 28,005 out of 30,793 successfully sequenced samples met the criteria to be deposited in the global GISAID database. During this period, we identified four variants of concern (VOC)-Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529). In detail, we observed 165 lineages in our dataset, with dominating Alpha, Delta and Omicron in three major consecutive incidence waves. This study aims to describe the results of a routine but high-level SARS-CoV-2 genomic surveillance program. Our study of SARS-CoV-2 genomes in collaboration with the Public Health Authority of the Slovak Republic also helped to inform the public about the epidemiological situation during the pandemic.

Conjunctival Swab Findings in 484 COVID-19 Patients in Four Hospital Centers in Slovakia.

Since 2020, the COVID-19 (Coronavirus Disease 2019) has quickly become a worldwide health problem. Ophthalmologists must deal with symptoms as well. For the positive detection in the conjunctival sac swab in COVID-19 patients hospitalized in Slovakia during March 2021 in four hospital centers, we used a test based on a polymerase chain reaction (PCR). In a group of 484 patients, 264 males (55%) and 220 females (45%) with clinical symptoms were identified with COVID-19 as a clinical diagnosis. The PCR test swab results from the conjunctival sac taken on the same day were positive in 58 patients (12%), 31 males (with a mean age of 74.6 ± 13.59 years) and 27 females (with a mean age of 70.63 ± 14.17 years); negative in 417 patients (86%); and 9 patients (2%) had an unclear result. The cycle threshold values comparing the nasopharynx and conjunctiva were also different in the group of all patients divided by age and gender. In COVID-19 patients the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detectable using PCR test in the nasopharynx but also in the conjunctival sac swab, where the positivity rate was only 12%.

Coronavirus disease (COVID-19) and conjunctival sac swab findings.

BACKGROUND: The purpose of this article is to evaluate the positivity of conjunctival sac swab by PCR (Polymerase chain reaction) test in COronaVIrus Disease 19 (COVID-19) patients. METHODS: Inclusion criteria of our study were COVID-19 patients hospitalized during March 2021 in inpatient wards at University Hospitals in towns Bratislava and Zilina, Slovakia. The conjunctival sac swabs collected by four ophthalmologists were stored for 24 h, then analyzed in the laboratory of the Department of Microbiology and Immunology, Jessenius Faculty of Medicine in Martin, Comenius University, Slovakia. The sampling apparatus, used for conjunctival sac swab, was the Dacron polyester swab. RESULTS: We examined one group of 302 COVID-19 patients, 168 Male (56%) and 134 Female (44%). The patients' mean age was 66.3 ± 13.66 years, ranging from 25 to 96 years, and the mean length of hospital stay in our patients with a nasopharyngeal positive PCR test was 7.33 ± 4.76, from 2 to 24 days. The PCR tests from the conjunctival sac swabs were positive in 33 patients (11%), negative in 259 patients (86%), and ten patients (3%) were with the unclear result. In the group of 33 positive patients were 17 males with a mean age of 74.6 ± 13.59 years and 16 females with a mean age of 70.63 ± 14.17 years. The cycle threshold (CT) values differed significantly between conjunctival sac swabs from the nasopharynx and the conjunctiva. Medians of the values were 25.1 (14.1, 32.1) and 31.5 (22.6, 36.6) (P <  0.001), respectively. CONCLUSION: This study affirmed that in COVID-19 patients the SARS-CoV-2 was detectable with PCR test in conjunctival sac swab, but the positivity rate was only about one to ten cases (11%).

Surveillance of SARS-CoV-2 lineage B.1.1.7 in Slovakia using a novel, multiplexed RT-qPCR assay.

The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia.

The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having a tremendous impact on the global economy, health care systems and the lives of almost all people in the world. The Central European country of Slovakia reached one of the highest daily mortality rates per 100,000 inhabitants in the first 3 months of 2021, despite implementing strong prophylactic measures, lockdowns and repeated nationwide antigen testing. The present study reports a comparison of the performance of the Standard Q COVID-19 antigen test (SD Biosensor) with three commercial RT-qPCR kits (vDetect COVID-19-MultiplexDX, gb SARS-CoV-2 Multiplex-GENERI BIOTECH Ltd. and Genvinset COVID-19 [E]-BDR Diagnostics) in the detection of infected individuals among employees of the Martin University Hospital in Slovakia. Health care providers, such as doctors and nurses, are classified as "critical infrastructure", and there is no doubt about the huge impact that incorrect results could have on patients. Out of 1231 samples, 14 were evaluated as positive for SARS-CoV-2 antigen presence, and all of them were confirmed by RT-qPCR kit 1 and kit 2. As another 26 samples had a signal in the E gene, these 40 samples were re-isolated and subsequently re-analysed using the three kits, which detected the virus in 22, 23 and 12 cases, respectively. The results point to a divergence not only between antigen and RT-qPCR tests, but also within the "gold standard" RT-qPCR testing. Performance analysis of the diagnostic antigen test showed the positive predictive value (PPV) to be 100% and negative predictive value (NPV) to be 98.10%, indicating that 1.90% of individuals with a negative result were, in fact, positive. If these data are extrapolated to the national level, where the mean daily number of antigen tests was 250,000 in April 2021, it points to over 4700 people per day being misinterpreted and posing a risk of virus shedding. While mean Ct values of the samples that were both antigen and RT-qPCR positive were about 20 (kit 1: 20.47 and 20.16 for Sarbeco E and RdRP, kit 2: 19.37 and 19.99 for Sarbeco E and RdRP and kit 3: 17.47 for ORF1b/RdRP), mean Ct values of the samples that were antigen-negative but RT-qPCR-positive were about 30 (kit 1: 30.67 and 30.00 for Sarbeco E and RdRP, kit 2: 29.86 and 31.01 for Sarbeco E and RdRP and kit 3: 27.47 for ORF1b/RdRP). It confirms the advantage of antigen test in detecting the most infectious individuals with a higher viral load. However, the reporting of Ct values is still a matter of ongoing debates and should not be conducted without normalisation to standardised controls of known concentration.