SARS-CoV-2 clade dynamics and their associations with hospitalisations during the first two years of the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic was characterised by rapid waves of disease, carried by the emergence of new and more infectious SARS-CoV-2 virus variants. How the pandemic unfolded in various locations during its first two years has yet to be sufficiently covered. To this end, here we are looking at the circulating SARS-CoV-2 variants, their diversity, and hospitalisation rates in Estonia in the period from March 2000 to March 2022. METHODS: We sequenced a total of 27,550 SARS-CoV-2 samples in Estonia between March 2020 and March 2022. High-quality sequences were genotyped and assigned to Nextstrain clades and Pango lineages. We used regression analysis to determine the dynamics of lineage diversity and the probability of clade-specific hospitalisation stratified by age and sex. RESULTS: We successfully sequenced a total of 25,375 SARS-CoV-2 genomes (or 92%), identifying 19 Nextstrain clades and 199 Pango lineages. In 2020 the most prevalent clades were 20B and 20A. The various subsequent waves of infection were driven by 20I (Alpha), 21J (Delta) and Omicron clades 21K and 21L. Lineage diversity via the Shannon index was at its highest during the Delta wave. About 3% of sequenced SARS-CoV-2 samples came from hospitalised individuals. Hospitalisation increased markedly with age in the over-forties, and was negligible in the under-forties. Vaccination decreased the odds of hospitalisation in over-forties. The effect of vaccination on hospitalisation rates was strongly dependent upon age but was clade-independent. People who were infected with Omicron clades had a lower hospitalisation likelihood in age groups of forty and over than was the case with pre-Omicron clades regardless of vaccination status. CONCLUSIONS: COVID-19 disease waves in Estonia were driven by the Alpha, Delta, and Omicron clades. Omicron clades were associated with a substantially lower hospitalisation probability than pre-Omicron clades. The protective effect of vaccination in reducing hospitalisation likelihood was independent of the involved clade.

Age-specific and genotype-specific carcinogenic human papillomavirus prevalence in a country with a high cervical cancer burden: results of a cross-sectional study in Estonia.

OBJECTIVES: To describe age-specific and type-specific carcinogenic human papillomavirus (HPV) prevalence prior to large-scale effect of HPV vaccines in Estonia and to analyse the risk factors associated with carcinogenic HPV. DESIGN: Cross-sectional study using self-administered questionnaire and self-collected vaginal swabs for detection of HPV infection. SETTING: Estonian Biobank database. PARTICIPANTS: Stratified random sample of women aged 30-33, 57-60 and 67-70 years living in one of the three largest counties in Estonia. Of 3065 women approached, 1347 (43.9%) returned questionnaires and specimens for HPV DNA detection. OUTCOME MEASURES: HPV prevalence and fully adjusted ORs with 95% CIs for risk factors. RESULTS: HPV prevalence was highest among women aged 30-33 years (18.7%; 95% CI 15.8 to 21.9) followed by those aged 67-70 years (16.7%; 95% CI 12.4 to 22.0) and 57-60 years (10.2%; 95% CI 7.8 to 13.3). HPV16 and HPV56 were the most common among women aged 30-33 years (both 4.0%; 95% CI 2.7 to 5.9), and HPV68 was the most common among women aged 57-60 years (2.8%; 95% CI 1.5 to 4.7) and 67-70 years (6.4%; 95% CI 3.6 to 10.4). Vaccination with nonavalent vaccine would have halved the carcinogenic HPV prevalence among women aged 30-33 years. The odds of infection with carcinogenic HPV were higher among women with six or more sexual partners among younger (OR 2.99; 95% CI 1.54 to 5.81) and older (OR 3.80; 95% CI 1.25 to 11.55) women and lower (OR 0.35; 95% CI 0.17 to 0.72) among younger married women. CONCLUSIONS: This study demonstrated U-shaped age-specific genotype profile of carcinogenic HPV prevalence, indicating that public health providers should focus on developing exit strategies for the cervical cancer screening programme in Estonia with a possible extension of HPV testing beyond the current screening age of 65 years. Generalisability of the findings of this study may be affected by the low response rate.

Novel Early Pregnancy Multimarker Screening Test for Preeclampsia Risk Prediction.

Preeclampsia (PE) is a common pregnancy-linked disease, causing preterm births, complicated deliveries, and health consequences for mothers and offspring. We have previously developed 6PLEX, a multiplex assay that measures PE-related maternal serum biomarkers ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 in a single test tube. This study investigated the potential of 6PLEX to develop novel PE prediction models for early pregnancy. We analyzed 132 serum samples drawn at 70-275 gestational days (g days) from 53 pregnant women (PE, n = 22; controls, n = 31). PE prediction models were developed using a machine learning strategy based on the stepwise selection of the most significant models and incorporating parameters with optimal resampling. Alternative models included also placental FLT1 rs4769613 T/C genotypes, a high-confidence risk factor for PE. The best performing PE prediction model using samples collected at 70-98 g days comprised of PTX3, sFlt-1, and ADAM12, the subject's parity and gestational age at sampling (AUC 0.94 [95%CI 0.84-0.99]). All cases, that developed PE several months later (onset 257.4 ± 15.2 g days), were correctly identified. The model's specificity was 80% [95%CI 65-100] and the overall accuracy was 88% [95%CI 73-95]. Incorporating additionally the placental FLT1 rs4769613 T/C genotype data increased the prediction accuracy to 93.5% [AUC = 0.97 (95%CI 0.89-1.00)]. However, 6PLEX measurements of samples collected at 100-182 g days were insufficiently informative to develop reliable PE prediction models for mid-pregnancy (accuracy <75%). In summary, the developed model opens new horizons for first-trimester PE screening, combining the easily standardizable 6PLEX assay with routinely collected antenatal care data and resulting in high sensitivity and specificity.

Value of soluble fms-like tyrosine kinase-1/placental growth factor test in third trimester of pregnancy for predicting preeclampsia in asymptomatic women.

OBJECTIVES: To estimate the value of screening maternal serum soluble fms-like tyrosine kinase/placental growth factor (sFlt-1/PlGF) ratio in asymptomatic women during 3rd trimester to predict preeclampsia (PE) development. METHODS: The investigated group comprised of 178 pregnant women. During this gestation, 24 cases had developed PE and 12 isolated gestational hypertension (GH); whereas 142 remained normotensive. Blood samples were collected between 180 and 259 gestational days (g.d.) when the participants were asymptomatic. Serums were analyzed using the BRAHMS sFlt-1 Kryptor/BRAHMS PlGF plus Kryptor PE ratio test (Thermo Fisher Scientific, Henningdorf, Germany). High-risk pregnancies for the PE development were defined as sFlt-1/PlGF>38. RESULTS: The detection rate (DR) for manifestation of PE≤30 days after sampling was 83.3% and overall DR during pregnancy 58.3%. Ten of 15 women having false positive prediction of PE suffered from GH, preterm birth and/or delivery of a small-for-gestational-age-newborn. False positive rate was significantly higher at 239-253 g.d. compared to sampling at 210-224 g.d. and 225-238 g.d. (21.9% vs. 7.8% and 5.3%; p < 0.05). CONCLUSIONS: The sFlt-1/PlGF test during 180-259 g.d. detected approximately half of subsequent PE cases. An optimal time to use the test for screening purposes was estimated 225-238 g.d. (DR 66.7%). False positive test results were more common to cases with other adverse pregnancy outcomes and samples drawn at higher gestational age.

C-allele of rs4769613 Near FLT1 Represents a High-Confidence Placental Risk Factor for Preeclampsia.

The variant rs4769613 T/C within the enhancer element near FLT1, an acknowledged gene in preeclampsia, was previously identified as a risk factor for preeclampsia in the genome-wide association study (GWAS) targeting placental genotypes. We aimed to test the robustness of this association in 2 Estonian cohorts. Both placental sample sets HAPPY PREGNANCY (Development of novel non-invasive biomarkers for fertility and healthy pregnancy; preeclampsia, n=44 versus nonpreeclampsia, n=1724) and REPROMETA (REPROgrammed fetal and/or maternal METAbolism; 52/277) exhibited suggestive association between rs4769613[C] variant and preeclampsia (logistic regression adjusted for gestational age and fetal sex, nominal P<0.05). Meta-analysis across 2 samples (96/2001) replicated the genome-wide association study outcome (Bonferroni corrected P=4×10-3; odds ratio, 1.75 [95% CI, 1.23-2.49]). No association was detected with gestational diabetes mellitus, preterm birth, and newborn parameters. Also, neither maternal nor paternal rs4769613 genotypes predisposed to preeclampsia. The exact role of placental rs4769613 genotype in the preeclampsia pathogenesis is to be clarified as no effect was detected on maternal baseline serum sFlt-1 (soluble fms-related receptor tyrosine kinase 1) levels. However, when placental FLT1 gene expression and maternal serum sFlt-1 measurements were stratified by placental rs4769613 genotypes, significantly higher transcript and biomarker levels were detected in preeclampsia versus nonpreeclampsia cases in the CC- and CT- (Student t test, P≤0.02), but not in the TT-genotype subgroup. We suggest that rs4769613 represents a conditional expression Quantitative Trait Locus, whereby only the enhancer with the C-allele reacts to promote the FLT1 expression in unfavorable placental conditions. The study highlighted that the placental FLT1 rs4769613 C-allele is a preeclampsia-specific risk factor. It may contribute to early identification of high-risk women, for example, when genotyped in the cffDNA available in maternal blood plasma.

Single-Tube Multimarker Assay for Estimating the Risk to Develop Preeclampsia.

BACKGROUND: Preeclampsia (PE) affects 2%-8% of all pregnancies worldwide. The predictive value of the currently used maternal serum fms-like tyrosine kinase-1/ placental growth factor (sFlt-1/PlGF) test is < 40% for PE onset within 4 weeks. We aimed to develop an innovative multiplex assay to improve PE prediction. METHODS: The 6PLEX assay combining the measurements of ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 was developed for the Luminex® xMAP platform. Assay performance was evaluated using 61 serum samples drawn from 53 pregnant women between 180 and 275 gestational days: diagnosed PE cases, n = 4; cases with PE onset within 4-62 days after sampling, n = 25; controls, n = 32. The B·R·A·H·M·S Kryptor sFlt-1/PlGF test (Thermo Fisher Scientific, Hennigsdorf, Germany) was applied as an external reference. Alternative PE prediction formulae combining 6PLEX measurements with clinical parameters were developed. RESULTS: There was a high correlation in sFlt-1/PlGF estimated for individual sera between the 6PLEX and B·R·A·H·M·S Kryptor immunoassays (Spearman's r = 0.93, P < 0.0001). The predictive power of the 6PLEX combined with gestational age and maternal weight at sampling reached AUC 0.99 (95% CI 0.97-1.00) with sensitivity 100.0% and specificity 96.9%. In all models, sFlt-1/PlGF derived from the B·R·A·H·M·S immunoassays exhibited the lowest AUC value (<0.87) and sensitivity (<80%) with broad confidence intervals (13%-92%). The estimated prognostic yield of the 6PLEX compared to the B·R·A·H·M·S assay was significantly higher (96.5% vs 73.7%; P = 0.0005). CONCLUSIONS: The developed single-tube multimarker assay for PE risk estimation in combination with clinical symptoms reached high prognostic yield (96.5%) and exhibited superior performance compared to the sFlt-1/PlGF test.

Application of Molecular Methods for Carbapenemase Detection.

This study has evaluated the correlation between different carbapenemases detection methods on carbapenem non-susceptible Klebsiella pneumoniae strains from Northern and Eastern Europe; 31 institutions in 9 countries participated in the research project, namely Finland, Estonia, Latvia, Lithuania, Russia, St. Petersburg, Poland, Belarus, Ukraine, and Georgia. During the research program, a total of 5,001 clinical K. pneumoniae isolates were screened for any carbapenem non-susceptibility by the disk diffusion method, Vitek 2 or Phoenix system following the EUCAST guideline on detection of resistance mechanisms, version 1.0. Strains isolated from outpatients and hospitalized patients from April 2015 to June 2015 were included. All types of samples (blood, pus, urine, etc.) excluding fecal screening or fecal colonization samples have been represented. In total, 171 carbapenemase screening-positive K. pneumoniae isolates (3.42%) were found and characterized. Several methods were used for detection of carbapenemases production, including Luminex assay (PCR and hybridization), whole genome sequencing, MALDI-TOF based Imipenem degradation assay, and immunochromatography testing. Minimal inhibitory concentration determination for Meropenem by agar-based gradient method was also used. Finally, 83 K. pneumoniae strains were carbapenemase negative by all confirmation methods (49.4% of all screening-positive ones), 74 - positive by three methods (44.0%), 8 - positive by two methods (4.8%) and 3 - positive by only one method (1.8%). The sensitivity of the tests was 96.3% for Whole genome sequencing and MALDI-TOF assay (both three undetected cases), and 95.1% for Luminex-Carba (4 undetected cases). The most commonly detected carbapenemases were NDM (n = 54) and OXA-48 (n = 26), followed by KPC-2, VIM-5, and OXA-72 (one case of each). Our results showed that different types of carbapenemases can be detected in the countries involved in the project. The sensitivity of our methods for carbapenemase detection (including screening as a first step and further confirmation tests) was >95%, but we would recommend using different methods to increase the sensitivity of detection and make it more precise.

Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.

Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability.