When transplant patients receive a new organ, it must be monitored for organ health, and the standard of care is typically an organ biopsy. Several studies have looked at using cfDNA biomarkers to monitor the health of the transplanted organ since a blood draw is less invasive than a biopsy; healthy organs release less cfDNA than failing organs and this can be measured by observing changes in the ratio of alleles at a site of genetic difference between the donor and host. While cfDNA fragments tend to be 167 bp or multiples thereof (the size required to wrap around a histone), fragments from transplanted organs are smaller and are present within 80-120 bp fragment size range (Pedini et. al., 2023). Working with such small DNA fragments requires optimized methods to both capture and amplify the DNA. Traditional DNA extraction methods have a lower fragment size cutoff of ~50 bp, and yield is typically lower as fragment size gets closer to 50 bp. PCR reactions must also be optimized for smaller amplicon size in order to amply small size DNA. This study will compare the recovery of different extraction methods in this size range. Differentiation of host and donor DNA can also be difficult; several studies have looked single nucleotide polymorphisms (SNPs) that vary across populations and may provide a site for differences in host and donor genomes. We utilized PCR assays designed by Kokelj et al. (2021) to evaluate the workflow's ability to capture and concentrate both spiked cfDNA and transplant patient cfDNA as a proof of concept for the detection of these biomarkers from donor organ plasma.

Read the entire article Introduction:  Although wastewater-based epidemiology (WBE) successfully functioned as a tool for monitoring the coronavirus disease 2019 (COVID-19) pandemic globally, relatively little is known about its utility in low-income countries. This study aimed to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater, estimate the number of infected individuals in the catchment areas, and correlate the results with the clinically reported COVID-19 cases in Addis Ababa, Ethiopia. Methods: A total of 323 influent and 33 effluent wastewater samples were collected from three Wastewater Treatment Plants (WWTPs) using a 24-h composite Moore swab sampling method from February to November 2023. The virus was captured using Ceres Nanotrap® Enhancement Reagent 2 and Nanotrap® Microbiome A Particles, and then nucleic acids were extracted using the Qiagen QIAamp Viral RNA Mini Kit. The ThermoFisher TaqPath™ COVID-19 kit was applied to perform real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to quantify the SARS-CoV-2 RNA. Wastewater viral concentrations were normalized using flow rate and number of people served. In the sampling period, spearman correlation was used to compare the SARS-CoV-2 target gene concentration to the reported COVID-19 cases. The numbers of infected individuals under each treatment plant were calculated considering the target genes’ concentration, the flow rate of treatment plants, a gram of feces per person-day, and RNA copies per gram of feces. Results: SARS-CoV-2 was detected in 94% of untreated wastewater samples. All effluent wastewater samples ( n  = 22) from the upflow anaerobic sludge blanket (UASB) reactor and membrane bioreactor (MBR) technology were SARS-COV-2 RNA negative. In contrast, two out of 11 effluents from Waste Stabilization Pond were found positive. Positive correlations were observed between the weekly average SARS-CoV-2 concentration and the cumulative weekly reported COVID-19 cases in Addis Ababa. The estimated number of infected people in the Kality Treatment catchment area was 330 times the number of COVID-19 cases reported during the study period in Addis Ababa. Discussion: This study revealed that SARS-CoV-2 was circulating in the community and confirmed previous reports of more asymptomatic COVID-19 cases in Ethiopia. Additionally, this study provides further evidence of the importance of wastewater-based surveillance in general to monitor infectious diseases in low-income settings. Conclusion: Wastewater-based surveillance of SARS-CoV-2 can be a useful method for tracking the increment of COVID-19 cases before it spreads widely throughout the community.

Read the article: https://journals.asm.org/doi/10.1128/spectrum.00689-23 ABSTRACT: This study reports development and optimization of a new method for the assessment and verification of the inactivation of peste des petits ruminants virus (PPRV) by chemical agents, including Triton X-100 and commercially available viral lysis buffers. Virus inactivation was confirmed by virus isolation (VI) on Vero cells following capture of the potential residual viruses from treated samples using Nanotrap magnetic virus particles (NMVPs). Since chemical agents are cytotoxic, treated PPRV samples could not be used directly for VI on Vero cell monolayers; instead, they were diluted in Eagle’s Minimum Essential Medium (EMEM) to neutralize cytotoxicity and then subjected to virus capture using NMVPs. The NMVPs and the captured viruses were then clarified on a magnetic stand, reconstituted in EMEM, and inoculated onto Vero cells that were examined for cytopathic effect (CPE). No CPE was observed on cells inoculated with treated viruses captured by NMVPs; but CPE was observed on cells inoculated with untreated viruses, including those captured by NMVPs. For further verification, the supernatants of the VI cultures (treated or untreated) were subjected to RNA extraction and PPRV-specific real-time RT-PCR (RT-qPCR). The cycle threshold values were undetectable for the supernatants of VI cultures inoculated with NMVPs reconstituted from treated PPRV but detectable for the supernatants of VI cultures inoculated with untreated PPRV or the NMVPs reconstituted from untreated PPRV, indicating complete inactivation of PPRV. This new method of verification of virus inactivation using NMVPs can be applied to other high impact viruses of agricultural or public health importance. Das A, Ahmed Z, Xu L, Jia W.2023.Assessment and verification of chemical inactivation of peste des petits ruminants virus by virus isolation following virus capture using Nanotrap magnetic virus particles. Microbiol Spectr11:e00689-23. https://doi.org/10.1128/spectrum.00689-23

Overview Using the Nanotrap® Microbiome B Particles to concentrate bacteria from wastewater samples from two rural wastewater treatment plants (WWTP), the Hawaii State Department of Health (HDOH) State Laboratories Division (SLD) performed studies of both temporal distribution and genetic diversity of AMR genes found within two Hawaiian communities. Results Nanotrap Microbiome B Particles were capable of concentrating bacteria containing each of the AMR targets at different points throughout the 13-week period (Fig 1). Within this period, KPC-1 and OXA-48 genes were prevalent within wastewater samples from both WWTPs, with the highest genomic presence of KPC-1. The NDM and IMP-1 genes were consistently detected from WWTP 1, while they were less detectable from WWTP 2 over the period. VIM-1 was detected from both WWTPs at low concentrations, and different points throughout the period. When the same samples were sequenced, the data showed large abundance values for both the KPC and OXA beta-lactamase genes, correlating with the dPCR data. NDM, IMP and VIM beta-lactamase genes were observed across the two WWTPs, but at much lower abundance. Wastewatern two separate Hawaiian islands. from the two WWTPs was found to show a similar AMR genetic diversity while being located on two separate Hawaiian islands.

Read the article: https://ascpt.onlinelibrary.wiley.com/doi/10.1002/cpt.3416 Abstract: BLOODPAC is a public-private consortium that develops best practices, coordinates clinical, and translational research, and manages the BLOODPAC Data Commons to support the liquid biopsy research community. BLOODPAC previously recommended 11 preanalytical Minimal Technical Data Elements (MTDEs) for BLOODPAC-sponsored studies and data submitted to BLOODPAC Data Commons ( Figure 1 ). The current landscape analysis evaluates the overlap of the MTDEs with 10 current best practices and standards documents related to clinical and research liquid biopsy applications.

ABSTRACT: Methods of wastewater concentration (electronegative filtration (ENF) versus magnetic bead-based concentration (MBC)) were compared for the analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), beta-2 micro-globulin, and human coronavirus OC43. Using ENF as the concentration method, two quantitative polymerase chain reaction (qPCR) analytical methods were also compared: volcano second generation (V2G)-qPCR and reverse transcriptase (RT)-qPCR measuring three different targets of the virus responsible for the COVID-19 illness (N1, modified N3, and ORF1ab). Correlations between concentration methods were strong and statistically significant for SARS-CoV-2 (r = 0.77, p < 0.001) and B2M (r = 0.77, p < 0.001). Comparison of qPCR analytical methods indicates that, on average, each method provided equivalent results with average ratios of 0.96, 0.96, and 1.02 for N3 to N1, N3 to ORF1ab, and N1 to ORF1ab and were supported by significant (p < 0.001) correlation coefficients (r = 0.67 for V2G (N3) to RT (N1), r = 0.74 for V2G (N3) to RT (ORF1ab), r = 0.81 for RT (N1) to RT (ORF1ab)). Overall results suggest that the two concentration methods and qPCR methods provide equivalent results, although variability is observed for individual measurements. Given the equivalency of results, additional advantages and disadvantages, as described in the discussion, are to be considered when choosing an appropriate method. Read the article: https://pubs.acs.org/doi/10.1021/acsestwater.2c00047?ref=pdf Comparison of Electronegative Filtration to Magnetic Bead-Based Concentration and V2G-qPCR to RT-qPCR for Quantifying Viral SARS-CoV-2 RNA from Wastewater Kristina M. Babler, Ayaaz Amirali, Mark E. Sharkey, Sion L. Williams, Melinda M. Boone, Gabriella A. Cosculluela, Benjamin B. Currall, George S. Grills, Jennifer Laine, Christopher E. Mason, Brian D. Reding, Stephan C. Schürer, Mario Stevenson, Dušica Vidović, and Helena M. Solo-Gabriele ACS ES&T Water 2022 2 (11), 2004-2013 DOI: 10.1021/acsestwater.2c00047

Wastewater testing has emerged as an effective tool for monitoring levels of SARS-CoV-2 infection in sewered communities. As of July 2024, PCR-based methods continue to be the most widely used methods in wastewater surveillance (1–3) . Data from PCR-based wastewater testing is usually available to public health authorities in near real time, typically within 5 to 7 days after waste enters the sewer (4,5) . Unfortunately, while these methods can accurately detect and quantify SARS-CoV-2, they are not usually used to differentiate between the multitude of variants, including variants that are classified as Variants of High Consequence (VOHC) and Variants of Concern (VOC) (6) . Currently, to identify these variants, the extracted nucleic acids must be analyzed using resource-intensive sequencing-based methods. Moreover, not every lab has access to sequencing technology, so the availability of equipment and expertise is also a roadblock. These costly and time-consuming sequencing methods, while informative, diminish some of the early warning benefits provided by wastewater surveillance. Moreover, not every lab has access to sequencing technology, creating additional barriers due to the availability of equipment and expertise. In response to these analytical shortcomings, we developed and assessed an alternative approach for variant monitoring in wastewater using customizable dPCR-based genotyping assays. This approach is an expansion from a previously described method for analyzing clinical samples utilizing customizable qPCR-based genotyping. Relative to sequencing, this approach is cost-effective, fast, and easily implemented.  We combined the dPCR-based wastewater genotyping approach along with the well-established NanotrapⓇ Particles virus concentration method as part of a wastewater processing protocol to perform SARS-CoV-2 genotyping in five wastewater testing labs across multiple regions in the United States. The results for the wastewater genotyping approach are displayed on a public-facing dashboard alongside clinical genotyping results and GISAID data (see https://tracker.rosalind.bio ). Despite genotyping fewer wastewater samples, our approach effectively detected signals of emerging variants and trends in SARS-CoV-2 variants within the community, similar to clinical analyses. For instance, in Georgia, the rapid rise and dominance of the Unknown and BA.2.86*/JN* variants in early 2024 were consistently observed in wastewater samples and closely matched trends in the GISAID clinical sequencing database. Similarly, the EG.5* and FL* variants showed elevated signals in wastewater before clinical detection, highlighting the early warning potential of wastewater testing. Detailed analysis of multiple datasets from various states revealed consistency in the rise and fall of variants across wastewater genotyping, clinical genotyping, and GISAID data. This consistency demonstrates that the prevalence of variants in wastewater closely matches that in clinical settings, underscoring the capability of wastewater-based surveillance to provide extended monitoring of circulating variants, often preceding clinical detections by several weeks. We further assessed the wastewater genotyping approach by calculating positive percent agreement for detection of four variants (JN, EG.5, FL, and XBB) between the genotyping results and whole genome sequencing results for a set of 129 matched samples that were analyzed using both methods. The agreement ranged between 54% agreement for FL to 97% agreement for JN, with an average of 76% agreement across all samples for all four variants. Additionally, we estimate that collecting and analyzing data using the dPCR genotyping method is significantly less expensive and time-consuming compared to next-generation sequencing. Labs that outsource next-generation sequencing face much higher costs and longer delays. Transitioning to multiplex dPCR for variant detection could further reduce both cost and turnaround time. Finally, we discuss the challenges and lessons learned in the development, validation, and implementation of dPCR-based wastewater genotyping. These findings support the use of wastewater-based surveillance as a complementary approach to clinical surveillance, offering a broader and more inclusive picture of variant prevalence and transmission in the community. SKU 44XXX SKU 10XXX

The study objective is to detect and quantify the proportion of SARS-CoV-2 variants in wastewater utilizing a genotyping assay designed by Applied Biosystems™ TaqMan™ SARS-CoV-2 Mutation Research Panel on either Digital PCR or Real-time qPCR platforms. Over eight assays and more than two hundred wastewater samples were evaluated using both Real-Time qPCR and Digital PCR as part of validation process. Genotyping assays were able to identify variant transition phases in wastewater. The study is funded by the National Institute of Health with collaboration between ROSALIND, Inc., Ceres Nanosciences, Inc., and Emory University. Emory University initiated the application of the Thermo Fisher SARS-CoV-2 variant detection assays to wastewater samples and developed this SOP. Ceres reviewed and revised the SOP. Study Protocol SKU 44XXX SKU 10XXX

Candida auris is a worldwide opportunistic fungus that can colonize in many areas throughout the human body, including blood and skin tissue, and that is also shed in stool samples. It has continually been declared an urgent threat by the Centers for Disease Control and Prevention (CDC). It has limited treatment options due to its persistence within healthcare settings, as well as the fact that it exhibits strong antifungal drug resistance. Surveillance of wastewater at a community level can increase preparedness for the treatment of C. auris infections by aiding in the identification of potential antimicrobial resistance genes present within this pathogen in a community. In this application note, see how to concentrate and extract C. auris and C. lusitaniae within a wastewater sample using a robust and high-throughput method. APPLICATION NOTE SKU 10XXX SKU 44XXX

Nature Communications 23 May 2024 / Nat Commun 15, 4391 Read the entire article Abstract Human immunodeficiency virus type-1 (HIV-1) is responsible for significant mortality and morbidity worldwide. Despite complete control of viral replication with antiretrovirals, cells with integrated HIV-1 provirus can produce viral transcripts. In a cross-sectional study of 84 HIV+ individuals of whom 43 were followed longitudinally, we found that HIV-1 RNAs are present in extracellular vesicles (EVs) derived from cerebrospinal fluid and serum of all individuals. We used seven digital droplet polymerase chain reaction assays to evaluate the transcriptional status of the latent reservoir. EV-associated viral RNA was more abundant in the CSF and correlated with neurocognitive dysfunction in both, the cross-sectional and longitudinal studies. Sequencing studies suggested compartmentalization of defective viral transcripts in the serum and CSF. These findings suggest previous studies have underestimated the viral burden and there is a significant relationship between latent viral transcription and CNS complications of long-term disease despite the adequate use of antiretrovirals.

Wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 has rapidly expanded across the globe as data has shown that WBE trends correlate with clinical case trends. The success of WBE with SARS-CoV-2 has led to interest in monitoring additional pathogens, including eukaryotes such as parasitic organisms and multi-drug resistant yeast. The CDC has identified several of these pathogens as emerging threats including Candida auris and several infection-causing parasitic organisms. In this poster, we describe how we use the Nanotrap® Microbiome B Particles, a new, magnetic particle-based automation-friendly method to enable improved detection of eukaryotic pathogens such as Candida auris, Cryptosporidium parvum, and Giardia lamblia in wastewater samples.

In this application note, we show that Nanotrap® Microbiome A Particles with the MagMAX™ Wastewater Ultra Nucleic Acid Isolation Kit is compatible with the Clear Dx™ FlexPro: Wastewater (SARS-CoV-2) and leads to higher genome coverage than a competitor method. Wastewater-based epidemiology (WBE) increased in prevalence during the pandemic as a way to measure disease load in a community cheaply and easily. It can be used to track disease at both a species and variant level; PCR applications are commonly used to measure different species and known variants, whereas sequencing is required to detect novel variants. Accurate and complete sequencing from wastewater requires clean and concentrated nucleic acid samples, so it is very dependent on the quality of sample preparation. Concentration methods need to concentrate microbes without concentrating inhibitors that are present in the sample matrix, and extraction methods need to purify the nucleic acid enough for adequate enzymatic activity during library preparation. The Clear Dx FlexPro: Wastewater (SARS-CoV-2) WGS Reagent and Automation Bundle uses targeted sequencing to identify SARS-CoV-2 strains in a system that provides a fully automated workflow for library prep and sequencing.