Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

SARS-CoV-2 variant detection from wastewater: rapid spread of B.1.1.7 lineage in Hungary.

Wastewater-based epidemiology (WBE) is a recognised tool for tracking community transmission of COVID-19. From the second half of 2020, the emergence of new, highly infective, more pathogenic or vaccine-escape SARS-CoV-2 variants is the major public health concern. Variant analysis in sewage might assist the early detection of new mutations. Weekly raw sewage samples from 22 wastewater treatment plants (WWTPs) in Hungary (representing 40% of the population) were analysed between December 2020 and March 2021 for signature mutations N501Y and del H69/V70 of B.1.1.7 lineage by melting point genotyping and RT-digital droplet PCR (RT-ddPCR). The latter method proved to be more efficient in parallel detection of different variants and also provides quantitative information. Wastewater surveillance indicated that the B.1.1.7 variant first emerged in Budapest in early January 2021 and rapidly became dominant in the entire country. Results are in close agreement with the available clinical data (Pearson's correlation coefficient, R = 0.9153). RT-ddPCR was confirmed to be a reliable tool for tracking emerging variant ratios in wastewaters. It is a rapid and cost-effective method compared to whole-genome sequencing, but only applicable for the detection of known mutations. Efficient variant surveillance might require the combination of multiple methods.

Topography, Spike Dynamics, and Nanomechanics of Individual Native SARS-CoV-2 Virions.

SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, displays a corona-shaped layer of spikes which play a fundamental role in the infection process. Recent structural data suggest that the spikes possess orientational freedom and the ribonucleoproteins segregate into basketlike structures. How these structural features regulate the dynamic and mechanical behavior of the native virion are yet unknown. By imaging and mechanically manipulating individual, native SARS-CoV-2 virions with atomic force microscopy, here, we show that their surface displays a dynamic brush owing to the flexibility and rapid motion of the spikes. The virions are highly compliant and able to recover from drastic mechanical perturbations. Their global structure is remarkably temperature resistant, but the virion surface becomes progressively denuded of spikes upon thermal exposure. The dynamics and the mechanics of SARS-CoV-2 are likely to affect its stability and interactions.

Tissue-Specific Accumulation and Isomerization of Valuable Phenylethanoid Glycosides from Plantago and Forsythia Plants.

A comparative phytochemical study on the phenylethanoid glycoside (PhEG) composition of the underground organs of three Plantago species (P. lanceolata, P. major, and P. media) and that of the fruit wall and seed parts of Forsythia suspensa and F. europaea fruits was performed. The leaves of these Forsythia species and six cultivars of the hybrid F. × intermedia were also analyzed, demonstrating the tissue-specific accumulation and decomposition of PhEGs. Our analyses confirmed the significance of selected tissues as new and abundant sources of these valuable natural compounds. The optimized heat treatment of tissues containing high amounts of the PhEG plantamajoside (PM) or forsythoside A (FA), which was performed in distilled water, resulted in their characteristic isomerizations. In addition to PM and FA, high amounts of the isomerization products could also be isolated after heat treatment. The isomerization mechanisms were elucidated by molecular modeling, and the structures of PhEGs were identified by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) techniques, also confirming the possibility of discriminating regioisomeric PhEGs by tandem MS. The PhEGs showed no cytostatic activity in non-human primate Vero E6 cells, supporting their safe use as natural medicines and allowing their antiviral potency to be tested.

ICTV Virus Taxonomy Profile: Filoviridae.

Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15-19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae.

Manipulating Active Structure and Function of Cationic Antimicrobial Peptide CM15 with the Polysulfonated Drug Suramin: A Step Closer to in Vivo Complexity.

Antimicrobial peptides (AMPs) kill bacteria by targeting their membranes through various mechanisms involving peptide assembly, often coupled with disorder-to-order structural transition. However, for several AMPs, similar conformational changes in cases in which small organic compounds of both endogenous and exogenous origin have induced folded peptide conformations have recently been reported. Thus, the function of AMPs and of natural host defence peptides can be significantly affected by the local complex molecular environment in vivo; nonetheless, this area is hardly explored. To address the relevance of such interactions with regard to structure and function, we have tested the effects of the therapeutic drug suramin on the membrane activity and antibacterial efficiency of CM15, a potent hybrid AMP. The results provided insight into a dynamic system in which peptide interaction with lipid bilayers is interfered with by the competitive binding of CM15 to suramin, resulting in an equilibrium dependent on peptide-to-drug ratio and vesicle surface charge. In vitro bacterial tests showed that when CM15⋅suramin complex formation dominates over membrane binding, antimicrobial activity is abolished. On the basis of this case study, it is proposed that small-molecule secondary structure regulators can modify AMP function and that this should be considered and could potentially be exploited in future development of AMP-based antimicrobial agents.

Taxonomy of the order Mononegavirales: second update 2018.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Imported Zika virus infections in Hungary between 2016 and 2018.

Zika virus is a mosquito-borne flavivirus with significant public health concern due to its association with neurological symptoms and intrauterine malformations. Although it is endemic in tropical and subtropical areas, sexual transmission raises the possibility of autochthonous spreading elsewhere. We describe the first laboratory diagnosed imported Zika-infections of Hungary, to highlight the challenges of microbiological identification of the pathogen, caused by serological cross-reactivity and short viremia. Serological examination was carried out using indirect immunofluorescent assay and enzyme-linked immunosorbent assay. Plaque-reduction neutralization test was used for verification purposes. A wide range of clinical specimens: serum, whole-blood, urine, saliva, and semen were analyzed by molecular methods, and sequencing was applied in case of PCR positive results to identify the virus strain. Zika-infected patients with previous vaccination against flaviviruses or possible flavivirus infection in the past showed high serological cross-reactivity, and even cross-neutralizing antibodies were observed. Zika virus RNA could be detected in urine specimen in case of two patients, and in EDTA-anticoagulated whole-blood sample of one patient. The detected strains belong to the Asian lineage of the virus. We presume that serological investigation of imported Zika virus could be altered by infections, vaccination of endemic flaviviruses in Hungary and vice versa.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Field Evaluation of Capillary Blood Samples as a Collection Specimen for the Rapid Diagnosis of Ebola Virus Infection During an Outbreak Emergency.

BACKGROUND: Reliable reverse transcription polymerase chain reaction (RT-PCR)-based diagnosis of Ebola virus infection currently requires a blood sample obtained by intravenous puncture. During the current Ebola outbreak in Guinea, we evaluated the usability of capillary blood samples collected from fingersticks of patients suspected of having Ebola virus disease (EVD) for field diagnostics during an outbreak emergency. METHODS: A total of 120 venous and capillary blood samples were collected from 53 patients admitted to the Ebola Treatment Centre in Guéckédou, Guinea, between July and August 2014. All sample specimens were analyzed by RT-PCR using the RealStar Filovirus Screen RT-PCR Kit 1.0 from altona Diagnostics (Germany). We compared samples obtained by venipuncture and those obtained by capillary blood sampling absorbed onto swab devices. RESULTS: The resulting sensitivity and specificity of tests performed with capillary blood samples were 86.8% (95% confidence interval [CI], 71.9%-95.6%; 33/38 patients) and 100% (95% CI, 84.6%-100%; 22/22 patients), respectively. CONCLUSIONS: Our data suggest that capillary blood samples could serve as an alternative to venous blood samples for the diagnosis of EVD in resource-limited settings during a crisis. This can be of particular advantage in cases when venipuncture is difficult to perform-for example, with newborns and infants or when adult patients reject venipuncture for cultural or religious reasons.

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial.

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.

First Broad-Range Serological Survey of Crimean-Congo Hemorrhagic Fever among Hungarian Livestock.

(1) Background: Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease endemic in Africa, Asia, the Middle East, and the Balkan and Mediterranean regions of Europe. Although no human CCHF cases have been reported, based on vector presence, serological evidence among small vertebrates, and the general human population, Hungary lies within high evidence consensus for potential CCHF introduction and future human infection. Thus, the aim of our pilot serosurvey was to assess CCHF seropositivity among cattle and sheep as indicator animals for virus circulation in the country. (2) Methods: In total, 1905 serum samples taken from free-range cattle and sheep in 2017 were tested for the presence of anti-CCHF virus IgG antibodies using commercial ELISA and commercial and in-house immunofluorescent assays. (3) Results: We found a total of eleven reactive samples (0.58%) from five administrative districts of Hungary comprising 8 cattle and 3 sheep. The most affected regions were the south-central and northwestern parts of the country. (4) Conclusions: Based on these results, more extended surveillance is advised, especially in the affected areas, and there should be greater awareness among clinicians and other high-risk populations of the emerging threat of CCHF in Hungary and Central Europe.

A novel ACE2 decoy for both neutralization of SARS-CoV-2 variants and killing of infected cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Evaluating the field performance of multiple SARS-Cov-2 antigen rapid tests using nasopharyngeal swab samples.

The SARS-CoV-2 pandemic, which started in December 2019, has been posing significant challenges to the health care system worldwide. As the pandemic spreads with rapidly increasing number of positive cases, early diagnosis of infected patients is crucial to successfully limit the spread of the virus. Although the real-time reverse-transcription polymerase chain reaction (RT-qPCR) is the recommended laboratory method to diagnose COVID-19 infection, many factors such as availability of laboratory equipment, reagents and trained personnel affect the use of time-consuming molecular techniques. To facilitate on-the-spot diagnosis of COVID-19, SARS-CoV-2 rapid antigen tests were developed by several different manufacturers. The evaluation of such rapid tests is particularly important due to the recent unanimous agreement by the European Commission Member States on a recommendation setting out a framework for the use of antigen rapid tests that contains a list of the mutually recognized assays and the basis of independent validation protocols. To evaluate the on-field performance of ten commercially available SARS-CoV-2 antigen rapid tests (CLINITEST Rapid COVID-19 Antigen Test, GenBody COVID-19 Antigen Test, GENEDIA W COVID-19 Ag Test, Healgen Coronavirus Antigen Rapid Test, Humasis COVID-19 Ag Test, VivaDiag SARS-CoV-2 Ag Rapid Test, Helix i-SARS-CoV-2 Ag Rapid Test, Roche SARS-CoV-2 Rapid Antigen Test, Abbot COVID-19 Ag Rapid Test and Vazyme SARS-CoV-2 Antigen Detection Kit) and compare with RT-qPCR as a reference method, the Hungarian National Public Health Center provided 1,597 antigen rapid tests to the National Ambulance Service, COVID-testing trucks and two hospitals treating COVID-19 patients. Sensitivity, specificity and accuracy were determined by performing the rapid test directly from nasopharyngeal swab samples of symptomatic individuals. For strongly positive samples (Ct < 25) sensitivities ranged between 66.7% and 100%, while for positive samples (Ct < 30) they gave a maximum sensitivity of 87.5%. The specificity of the tests was ranging between 79% to 100%. The results presented here are of high importance to the European Commission and also help governmental decision-making regarding the application of the proper rapid tests for screening different at-risk populations. Nonetheless, SARS-Cov-2 rapid tests play an important role in early and on-the-spot diagnosis of potentially infected individuals.

In Vitro Determination of Inhibitory Effects of Humic Substances Complexing Zn and Se on SARS-CoV-2 Virus Replication.

(1) Background: Humic substances are well-known human nutritional supplement materials and they play an important performance-enhancing role as animal feed additives. For decades, ingredients of humic substances have been proven to carry potent antiviral effects against different viruses. (2) Methods: Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern ("Alpha Variant") in a VeroE6 cell line. (3) Results: This combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. (4) Conclusions: Even picomolar concentration ranges of humic substances, Vitamin C and Zn/Se ions in the given composition, were enough to achieve 50% viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.

Nationwide effectiveness of five SARS-CoV-2 vaccines in Hungary-the HUN-VE study.

OBJECTIVES: The Hungarian vaccination campaign was conducted with five different vaccines during the third wave of the coronavirus disease 2019 (COVID-19) pandemic in 2021. This observational study (HUN-VE: Hungarian Vaccine Effectiveness) estimated vaccine effectiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and COVID-19-related mortality in 3.7 million vaccinated individuals. METHODS: Incidence rates of SARS-CoV-2 infection and COVID-19-related mortality were calculated using data from the National Public Health Centre surveillance database. Estimated vaccine effectiveness was calculated as 1 - incidence rate ratio ≥7 days after the second dose for each available vaccine versus an unvaccinated control group using mixed-effect negative binomial regression controlling for age, sex and calendar day. RESULTS: Between 22 January 2021 and 10 June 2021, 3 740 066 Hungarian individuals received two doses of the BNT162b2 (Pfizer-BioNTech), HB02 (Sinopharm), Gam-COVID-Vac (Sputnik-V), AZD1222 (AstraZeneca), or mRNA-1273 (Moderna) vaccines. Incidence rates of SARS-CoV-2 infection and COVID-19-related death were 1.73-9.3/100 000 person-days and 0.04-0.65/100 000 person-days in the fully vaccinated population, respectively. Estimated adjusted effectiveness varied between 68.7% (95% CI 67.2%-70.1%) and 88.7% (95% CI 86.6%-90.4%) against SARS-CoV-2 infection, and between 87.8% (95% CI 86.1%-89.4%) and 97.5% (95% CI 95.6%-98.6%) against COVID-19-related death, with 100% effectiveness in individuals aged 16-44 years for all vaccines. CONCLUSIONS: Our observational study demonstrated the high or very high effectiveness of five different vaccines in the prevention SARS-CoV-2 infection and COVID-19-related death.

Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation.

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%-10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

New geographical area on the map of Crimean-Congo hemorrhagic fever virus: First serological evidence in the Hungarian population.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease that is endemic in Africa, Asia, the Middle East, and the Balkan region of Europe; the disease is spreading northwards following widespread distribution of the main vector, Hyalomma marginatum, which was first found in Hungary in 2011. The aim of this pilot sero-surveillance study was to assess CCHF seroprevalence in Hungary. A total of 2700 serum samples obtained from healthy volunteer blood donors were screened using an in-house immunofluorescence assay and a commercially available ELISA kit. We found ten (0.37 %) seropositive donors. The western and central regions proved to be the most affected areas, with a prevalence of 2.97 %. Higher positivity was found among male donors (0.55 %) and younger donors (18-34 years; 0.78 %). Based on these results, a more extended surveillance focusing on specific at-risk populations and animals is advised. The results should also raise the awareness of clinicians and other high-risk populations, such as foresters and hunters, about the emerging threat of CCHF in Hungary.

Ahead of the second wave: Early warning for COVID-19 by wastewater surveillance in Hungary.

Wastewater based epidemiology is a potential early warning tool for the detection of COVID-19 outbreak. Sewage surveillance for SARS-CoV-2 RNA was introduced in Hungary after the successful containment of the first wave of the pandemic to forecast the resurge of infections. Three wastewater treatment plants servicing the entire population (1.8 million) of the capital, Budapest were sampled weekly. 24 h composite (n = 44) and grab samples (n = 21) were concentrated by an in-house flat sheet membrane ultrafiltration method. The efficiency and reproducibility of the method was comparable to those previously published. SARS-CoV-2 RNA was quantified using RT-qPCR of the N gene. The first positive signal in sewage was detected 2 weeks before the rise in case numbers. Viral concentration and volume-adjusted viral load correlated to the weekly new cases from the same week and the rolling 7-day average of active cases in the subsequent week. The correlation was more pronounced in the ascending phase of the outbreak, data was divergent once case numbers plateaued. Wastewater surveillance was found to be effective in predicting the second wave of the outbreak in Hungary. Data indicated that even relatively low frequency (weekly) sampling is useful and at the same time, cost effective tool in outbreak detection.