Prediction of hospitalisations based on wastewater-based SARS-CoV-2 epidemiology.

Wastewater-based epidemiology is widely applied in Austria since April 2020 to monitor the SARS-CoV-2 pandemic. With a steadily increasing number of monitored wastewater facilities, 123 plants covering roughly 70 % of the 9 million population were monitored as of August 2022. In this study, the SARS-CoV-2 viral concentrations in raw sewage were analysed to infer short-term hospitalisation occupancy. The temporal lead of wastewater-based epidemiological time series over hospitalisation occupancy levels facilitates the construction of forecast models. Data pre-processing techniques are presented, including the approach of comparing multiple decentralised wastewater signals with aggregated and centralised clinical data. Time­lead quantification was performed using cross-correlation analysis and coefficient of determination optimisation approaches. Multivariate regression models were successfully applied to infer hospitalisation bed occupancy. The results show a predictive potential of viral loads in sewage towards Covid-19 hospitalisation occupancy, with an average lead time towards ICU and non-ICU bed occupancy between 14.8-17.7 days and 8.6-11.6 days, respectively. The presented procedure provides access to the trend and tipping point behaviour of pandemic dynamics and allows the prediction of short-term demand for public health services. The results showed an increase in forecast accuracy with an increase in the number of monitored wastewater treatment plants. Trained models are sensitive to changing variant types and require recalibration of model parameters, likely caused by immunity by vaccination and/or infection. The utilised approach displays a practical and rapidly implementable application of wastewater-based epidemiology to infer hospitalisation occupancy.

Wastewater surveillance of SARS-CoV-2 in Austria: development, implementation, and operation of the Tyrolean wastewater monitoring program.

Wastewater-based epidemiology (WBE) is an effective approach for tracking information on spatial distribution and temporal trends of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the community level. Herein, the development, implementation, and operation of the wastewater monitoring program serving Tyrol - a federal province of Austria - are described. The development of this program was initiated by Tyrolean health authorities at the end of the first phase of the Coronavirus disease 2019 (COVID-19) pandemic (May 2020). In close co-operation with the water sector and academic institutions, efficient and effective workflows and processes for wastewater surveillance were established. The monitoring program went into operation in November 2020. By the end of July 2021, a total of 5,270 wastewater influent samples collected at 43 sites were analyzed. The monitoring program provided valuable insights into the development of the pandemic situation in Tyrol and fulfilled several tasks that are of importance in different phases of the pandemic. It represented an early-warning system, provided independent confirmation of temporal trends in COVID-19 prevalence, enabled the assessment of the effectiveness of measures, alerted about bursts of disease activity, and provided evidence for the absence of COVID-19. These findings underline the importance of establishing national wastewater monitoring programs as a complementary source of information for efficient and effective pandemic management.

Viral variant-resolved wastewater surveillance of SARS-CoV-2 at national scale.

SARS-CoV-2 surveillance by wastewater-based epidemiology is poised to provide a complementary approach to sequencing individual cases. However, robust quantification of variants and de novo detection of emerging variants remains challenging for existing strategies. We deep sequenced 3,413 wastewater samples representing 94 municipal catchments, covering >59% of the population of Austria, from December 2020 to February 2022. Our system of variant quantification in sewage pipeline designed for robustness (termed VaQuERo) enabled us to deduce the spatiotemporal abundance of predefined variants from complex wastewater samples. These results were validated against epidemiological records of >311,000 individual cases. Furthermore, we describe elevated viral genetic diversity during the Delta variant period, provide a framework to predict emerging variants and measure the reproductive advantage of variants of concern by calculating variant-specific reproduction numbers from wastewater. Together, this study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without extensive individual monitoring.

Increased forensic efficiency of DNA fingerprints through simultaneous resolution of length and nucleotide variability by high-performance mass spectrometry.

Short tandem repeat (STR) typing is the most powerful method for determining the origin of a sample for a number of molecular disciplines such as medical genetics, population genetics, tumor analysis, transplantation medicine, or forensic crime scene analysis. STR alleles are routinely differentiated based upon their fragment size by electrophoresis under denaturing conditions, which does not take nucleotide variability into consideration. This simplification leads to loss of biological information as the nature of the individual sequence motifs that build an STR is not described. An alternative detection platform would be mass spectrometry, which captures the underlying sequence variation by comparing the molecular masses of DNA fragments. Here, we demonstrate that the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (ICEMS) is able to simultaneously detect length and nucleotide variability in STRs. Overall, 21 forensically relevant STRs that are also used in other scientific fields were screened in an Austrian population sample for the occurrence of nucleotide variability within or close to the repeat region. A total of 11 of the investigated loci (SE33, D2S1338, vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, and D2S441) brought additional allele (sequence) variants. Forensic efficiency, as determined by typical statistical parameters, was significantly increased by 20 to 30%. The beauty of ICEMS-STR-analysis is the fact that it represents one of the few technological advancements that allows direct comparison of newly generated data with existing data such as stored in DNA databases, which have a retarding effect on new developments.

A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA.

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs)--generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets--enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

Estimating the probability of identity in a random dog population using 15 highly polymorphic canine STR markers.

Dog DNA-profiling is becoming an important supplementary technology for the investigation of accident and crime, as dogs are intensely integrated in human social life. We investigated 15 highly polymorphic canine STR markers and two sex-related markers of 131 randomly selected dogs from the area around Innsbruck, Tyrol, Austria, which were co-amplified in three PCR multiplex reactions (ZUBECA6, FH2132, FH2087Ua, ZUBECA4, WILMSTF, PEZ15, PEZ6, FH2611, FH2087Ub, FH2054, PEZ12, PEZ2, FH2010, FH2079 and VWF.X). Linkage testing for our set of marker suggested no evidence for linkage between the loci. Heterozygosity (HET), polymorphism information content (PIC) and the probability of identity (P((ID)theoretical), P((ID)unbiased), P((ID)sib)) were calculated for each marker. The HET((exp))-values of the 15 markers lie between 0.6 (VWF.X) and 0.9 (ZUBECA6), P((ID)sib)-values were found to range between 0.49 (VWF.X) and 0.28 (ZUBECA6). Moreover, the P((ID)sib) was computed for sets of loci by sequentially adding single loci to estimate the information content and the usefulness of the selected marker sets for the identification of dogs. The estimated P((ID)sib) value of all 15 markers amounted to 8.5 x 10(-8). The presented estimations turned out to be a helpful approach for a reasonable choice of markers for the individualisation of dogs.