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Wastewater treatment plant (WWTP) influent sampling is commonly used in wastewater-based disease surveillance to assess the circulation of pathogens in the population aggregated in a catchment area. However, the signal can be lost within the sewer network due to adsorption, degradation, and dilution processes. The present work aimed to investigate the dynamics of SARS-CoV-2 concentration in three sub-catchments of the sewer system in the city of Hildesheim, Germany, characterised by different levels of urbanisation and presence/absence of industry, and to evaluate the benefit of sub-catchment sampling compared to WWTP influent sampling. Our study shows that sampling and analysis of virus concentrations in sub-catchments with particular settlement structures allows the identification of high concentrations of the virus at a local level in the wastewater, which are lower in samples collected at the inlet of the treatment plant covering the whole catchment. Higher virus concentrations per inhabitant were found in the sub-catchments in comparison to the inlet of the WWTP. Additionally, sewer sampling provides spatially resolved concentrations of SARS-CoV-2 in the catchment area, which is important for detecting local high incidences of COVID-19. [ABSTRACT FROM AUTHOR]

Background: There is a critical need for a rapid and sensitive pathogen detection method for septic patients. This study aimed to investigate the diagnostic efficacy of Digital droplet polymerase chain reaction (ddPCR) in identifying pathogens among suspected septic patients. Methods: We conducted a prospective pilot diagnostic study to clinically validate the multiplex ddPCR panel in diagnosing suspected septic patients. A total of 100 sepsis episodes of 89 patients were included in the study. Results: In comparison to blood culture, the ddPCR panel exhibited an overall sensitivity of 75.0% and a specificity of 69.7%, ddPCR yielded an additional detection rate of 17.0% for sepsis cases overall, with a turnaround time of 2.5 h. The sensitivity of ddPCR in the empirical antibiotic treatment and the non-empirical antibiotic treatment group were 78.6% versus 80.0% (p > 0.05). Antimicrobial resistance genes were identified in a total of 13 samples. Whenever ddPCR detected the genes beta-lactamase-Klebsiella pneumoniae carbapenemase (blaKPC) or beta-lactamase-New Delhi metallo (blaNDM), these findings corresponded to the cultivation of carbapenem-resistant gram-negative bacteria. Dynamic ddPCR monitoring revealed a consistent alignment between the quantitative ddPCR results and the trends observed in C-reactive protein and procalcitonin levels. Conclusions: Compared to blood culture, ddPCR exhibited higher sensitivity for pathogen diagnosis in suspected septic patients, and it provided pathogen and drug resistance information in a shorter time. The quantitative results of ddPCR generally aligned with the trends seen in C-reactive protein and procalcitonin levels, indicating that ddPCR can serve as a dynamic monitoring tool for pathogen load in septic patients. [ABSTRACT FROM AUTHOR]

Purpose of the study. To evaluate the cellular, genomic (gene copy number) and transcriptomic (gene expression) effects of P.hybridus (L.) secondary metabolites when they affect the HeLa cell line.Materials and methods. The isolation of secondary metabolites from plant material and its identification were carried out by preparative chromatography. The composition was determined using mass spectrometric analysis, and the final verification of structural formulas was carried out by nuclear magnetic resonance at the Department of Natural Compounds, the Faculty of Chemistry of the Southern Federal University. The subsequent phase of the study was conducted using both cultural and molecular methods. HeLa cells were cultivated under standard conditions in a MEM medium. Once the confluence level was reached 75–80 %, the nutrient medium was replaced with the introduction of the studied compounds (at a concentration of 4 micrograms/ml) and cultivated for 72 hours. Cell mortality was determined using a NanoEnTek JuliFl counter (Korea) in the presence of 0.4 % trypan blue. The assessment of apoptosis following secondary metabolite exposure was conducted on a BD FACSCanto II flow cytometer using the FITC Annexin V Apoptosis Detection Kit I. The level of replication and expression of the genes responsible for apoptosis was assessed by digital droplet PCR (ddPCR).Results. The following compounds were isolated and verified, and were assigned the following sequence numbers to facilitate their use in the experiment: No. 2 – 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one, No. 3 – 5-(hydroxymethyl) furan-2-carbaldehyde, No. 5.3 – 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, P. hybridus (L.) At the stage of cell death assessment, it was found that the greatest effect was achieved in the compound under ordinal No. 2. However, the evaluation of the copy number and expression of the CASP8, CASP9, CASP3, BAX, BCL2, TP53, MDM2, CDKN1B, CDK1, CCND1, CCND3, and RB1 genes by DD-PCR revealed the presence of apoptosis initiation in tumor cells at the molecular level under the action of compounds No. 2 and No. 5.3 obtained from P. hybridus (L.).Conclusion. The outcomes were multifeatured. Only compound 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one exhibited a pronounced cytostatic effect out of all compounds utilized in the experiment. Concurrently, the compound 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde was found to induce an increase in the expression of the CASP3, CASP8, TP53, and BAX genes.

Simple Summary: From detection to monitoring therapeutic response, circulating tumor DNA has emerged at the forefront of numerous clinical applications in oncology. It provides a non-invasive, real-time window into the evolving tumor as the cancer progresses. Advances in liquid biopsies and circulating tumor DNA analysis are enhancing their diagnostic applicability with tools like liposomal nanoparticles and antibody priming agents by binding to cell-free DNA in circulation and aiding in evading clearance mechanisms. The development of highly sensitive techniques such as digital droplet PCR and next-generation sequencing-based technologies has enabled the detection of minute ctDNA fractions amid high background, increasing its potential for early detection and minimal residual disease. Circulating tumor DNA (ctDNA), a fragment of tumor DNA found in the bloodstream, has emerged as a revolutionary tool in cancer management. This review delves into the biology of ctDNA, examining release mechanisms, including necrosis, apoptosis, and active secretion, all of which offer information about the state and nature of the tumor. Comprehensive DNA profiling has been enabled by methods such as whole genome sequencing and methylation analysis. The low abundance of the ctDNA fraction makes alternative techniques, such as digital PCR and targeted next-generation exome sequencing, more valuable and accurate for mutation profiling and detection. There are numerous clinical applications for ctDNA analysis, including non-invasive liquid biopsies for minimal residual disease monitoring to detect cancer recurrence, personalized medicine by mutation profiling for targeted therapy identification, early cancer detection, and real-time evaluation of therapeutic response. Integrating ctDNA analysis into routine clinical practice creates promising avenues for successful and personalized cancer care, from diagnosis to treatment and follow-up. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection. [ABSTRACT FROM AUTHOR]

ABSTRACT: Enteropathies are a serious concern in racing pigeons as they significantly impair performance in races and their training, and viruses are suspected to be one of the main factors. Astroviruses are well-known to be responsible for causing enteric disease in humans and various other animals including birds, although their prevalence and pathogenicity in pigeons is poorly understood. In this study, we investigated 2 groups of young racing pigeons (sick—study group and healthy—control group) to assess the correlation between the number of astrovirus genome copies in cloacal swabs and the occurrence of enteropathy. To determine this, we developed a novel TaqMan quantitative PCR (qPCR) and digital droplet PCR (ddPCR) methods for astrovirus detection and absolute quantitative analysis. We also performed high-throughput sequencing to obtain the complete genome sequences and establish the genetic similarity of the obtained strains to known astroviruses of poultry and other avian species. Two new complete genome sequences of pigeon astroviruses in the Avastrovirus genus were identified, representing 2 new species. These were found most closely related to astroviruses identified in Columbidae species and chickens. They share an average of 75.8% genome-wide pairwise identity and 57.6% and 64.6% capsid protein sequence identity with other unclassified columbid avastrovirus sequences in GenBank. Although the difference in prevalence of astrovirus in the study and control group was found statistically insignificant, there was a significant difference between the number of genome copies in positive samples from both groups. These unambiguous results leave the role of astroviruses as enteropathogenic factors in pigeons still undetermined.

To establish accurate detection methods of process‐specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific‐process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process‐specific enzyme‐linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non‐process‐specific commercial ELISA kit and the process‐specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process‐specific HCPs, could not be detected by the non‐process‐specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process‐specific ELISA has high sensitivity in detecting process‐specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future. Significance statement: Process‐specific immunological detection methods like a process‐specific double antibody sandwich enzyme‐linked immunosorbent assay method for HCP and a ddPCR absolute quantification method for rcDNA were established in this study, which provided a reference to improve the HCP and rcDNA detection system. It could be conducive to improving the process, improving the product quality, and achieving convergence with the standards of biopharmaceuticals in developed countries in Europe and America. [ABSTRACT FROM AUTHOR]

Flow cytometry (FCM) and quantitative PCR (qPCR) are conventional methods for assessing CAR-T expansion, while digital droplet PCR (ddPCR) is emerging as a promising alternative. We monitored CAR-T transcript expansion in 40 B-NHL patients post-infusion of CAR-T products (axi-cel; tisa-cel; and brexu-cel) with both His-Tag FCM and ddPCR techniques. Sensitivity and predictive capacity for efficacy and safety outcomes of ddPCR were analyzed and compared with FCM. A significant correlation between CAR-T counts determined by FCM and CAR transcripts assessed by ddPCR (p < 0.001) was observed. FCM revealed median CD3+CAR+ cell counts at 7, 14, and 30 days post-infusion with no significant differences. In contrast, ddPCR-measured median copies of CAR-T transcripts demonstrated significant lower copy numbers in tisa-cel recipients compared to the other products at day 7 and day 14. Patients with a peak of CAR transcripts at day 7 exceeding 5000 copies/microg gDNA, termed "good CAR-T expanders", were more likely to achieve a favorable response at 3 months (HR 10.79, 95% CI 1.16–100.42, p = 0.036). Good CAR-T expanders showed superior progression-free survival at 3, 6, and 12 months compared to poor CAR-T expanders (p = 0.088). Those reaching a peak higher than 5000 copies/microg gDNA were more likely to experience severe CRS and ICANS. DdPCR proves to be a practical method for monitoring CAR-T expansion, providing quantitative information that better predicts both treatment outcomes and toxicity. [ABSTRACT FROM AUTHOR]

Aims: The differential diagnosis of small hepatocellular nodules in cirrhosis between dysplastic nodules and hepatocellular carcinoma (HCC) remains challenging on biopsy. As TERT promoter (pTERT) mutations may indicate the nodules already engaged in the malignant process, the aim of this study was to identify histological criteria associated with pTERT mutations by detecting these mutations by ddPCR in small formalin‐fixed paraffin‐embedded (FFPE) hepatocellular nodules arising in cirrhosis. Methods and results: We built a bicentric cohort data set of 339 hepatocellular nodules < 2 cm from cirrhotic samples, divided into a test cohort of 299 resected samples and a validation cohort of 40 biopsies. Pathological review, based on the evaluation of 14 histological criteria, classified all nodules. pTERT mutations were identified by ddPCR in FFPE samples. Among the 339 nodules, ddPCR revealed pTERT mutations in 105 cases (31%), including 90 and 15 cases in the test and validation cohorts, respectively. On multivariate analysis, three histological criteria were associated with pTERT mutations in the test cohort: increased cell density (P = 0.003), stromal invasion (P = 0.036) and plate‐thickening anomalies (P < 0.001). With the combination of at least two of these major criteria, the AUC for predicting pTERT mutations was 0.84 in the test cohort (sensitivity: 86%, specificity: 83%) and 0.81 in the validation cohort (sensitivity: 87%, specificity: 76%). Conclusions: We identified three histological criteria as surrogate markers of pTERT mutations that may be used in routine biopsy to more clearly classify small hepatocellular nodules arising in cirrhosis. [ABSTRACT FROM AUTHOR]

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient–dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection. [ABSTRACT FROM AUTHOR]

Abstract Objective Endocrine therapy is frequently administered in patients with hormone dependent (HR+) metastatic endometrial cancer. ESR1 mutations have emerged as a key mechanism of aromatase inhibitor (AI) resistance in HR + metastatic breast cancer and can be monitored using circulating tumor DNA (ctDNA). The aim of this study was to explore the incidence and clinical relevance of circulating ESR1 mutations in patients treated by AI or megestrol acetate (M) for advanced endometrial carcinoma. Methodology This single-center retrospective study was performed at the Henri Becquerel Center (Rouen) and looked for circulating ESR1 gene mutations by droplet digital PCR (E380Q, L536R, Y537S, Y537N, Y537C, D538G, S463P) in patients with advanced HR + endometrial carcinoma treated between 2008 and 2020 for at least 30 days by AI or M. Analyses were performed before exposure and at progression/during endocrine therapy. Results Twenty-two patients were included: 13 were treated with AI, 12 of whom progressed; 9 patients were treated with M, 8 of whom progressed. 68.1% of the patients had low-grade endometrial carcinoma and 54.5% had received chemotherapy in the metastatic setting. The median duration of treatment was 152 days (min 47 – max 629) with AI and 155 days (min 91-max 1297) with M. Under AI, there was no ESR1 mutation at baseline, and one Y537C mutation at progression with a variant allele frequency (VAF) of 0.14%. Under M, one patient had a Y537C (VAF 0.2%) at baseline that disappeared during treatment. Another patient had a Y537S mutation emergence at progression after 91 days of treatment (VAF 1.83%). There was no significant difference between the circulating DNA concentration before and after hormone therapy (p = 0.16). Conclusion ESR1 mutations do not seem to be involved in the mechanisms of resistance to AI or M in HR+ endometrial cancer. The clinical relevance of their detection is not demonstrated.

Abstract The Second National Workshop on Environmental DNA was held on September 12–15, 2022, at the Southern California Coastal Water Research Project (SCCWRP) in Southern California and was focused on transitioning eDNA from research to management applications. The Workshop was attended by 150 people in‐person and an additional 200 more online. Workshop attendees represented a broad cross‐section of disciplines and backgrounds, including research scientists, state, and federal agencies, and those in the environmental management sector. This diverse collection of attendees assembled with the goal of achieving cross‐sector collaboration and working together to identify the necessary next steps to move eDNA methods into the management application mainstream. The Workshop structure included a Training Day oriented towards environmental managers and those new to eDNA science, to facilitate a common ground for discussions on subsequent days. The Plenary Day focused on case studies about eDNA applications and culminated with a roundtable panel discussion with local, state, and federal agency representatives on eDNA method readiness and the road to method adoption. Among the key takeaways from the Workshop was bridging the communication gap between researchers and managers because scientists often focus on technical details and the unknowns, giving the impression that eDNA science is not yet mature, whereas managers want to hear consensus statements about readiness and a roadmap for method adoption, including standard operating procedures, lab accreditation, and unified sequence libraries. This outcome was a clear directive for many scientists in attendance that it is time to stop letting perfect be the enemy of good and to focus future efforts on method harmonization and a national strategy towards method adoption. The Workshop concluded with a working session of invited participants to identify key priorities and needs to achieve the goals highlighted in the Workshop discussions.

Objective: Endocrine therapy is frequently administered in patients with hormone dependent (HR+) metastatic endometrial cancer. ESR1 mutations have emerged as a key mechanism of aromatase inhibitor (AI) resistance in HR + metastatic breast cancer and can be monitored using circulating tumor DNA (ctDNA). The aim of this study was to explore the incidence and clinical relevance of circulating ESR1 mutations in patients treated by AI or megestrol acetate (M) for advanced endometrial carcinoma. Methodology: This single-center retrospective study was performed at the Henri Becquerel Center (Rouen) and looked for circulating ESR1 gene mutations by droplet digital PCR (E380Q, L536R, Y537S, Y537N, Y537C, D538G, S463P) in patients with advanced HR + endometrial carcinoma treated between 2008 and 2020 for at least 30 days by AI or M. Analyses were performed before exposure and at progression/during endocrine therapy. Results: Twenty-two patients were included: 13 were treated with AI, 12 of whom progressed; 9 patients were treated with M, 8 of whom progressed. 68.1% of the patients had low-grade endometrial carcinoma and 54.5% had received chemotherapy in the metastatic setting. The median duration of treatment was 152 days (min 47 – max 629) with AI and 155 days (min 91-max 1297) with M. Under AI, there was no ESR1 mutation at baseline, and one Y537C mutation at progression with a variant allele frequency (VAF) of 0.14%. Under M, one patient had a Y537C (VAF 0.2%) at baseline that disappeared during treatment. Another patient had a Y537S mutation emergence at progression after 91 days of treatment (VAF 1.83%). There was no significant difference between the circulating DNA concentration before and after hormone therapy (p = 0.16). Conclusion: ESR1 mutations do not seem to be involved in the mechanisms of resistance to AI or M in HR+ endometrial cancer. The clinical relevance of their detection is not demonstrated. [ABSTRACT FROM AUTHOR]

There is no robust predictor of response to chemotherapy (CT) in unresectable pancreatic adenocarcinomas (UPA). The objective of the KRASCIPANC study was to analyze the kinetics of cell-free DNA (cfDNA)/circulating tumor DNA (ctDNA) as a predictor of response to CT in UPA. Blood samples were collected just before first CT and at day 28. The primary endpoint was the kinetics of KRAS -mutated ctDNA by digital droplet PCR between D0 and D28 as a predictor of progression-free survival (PFS). We analyzed 65 patients with a KRAS -mutated tumor. A high level of cfDNA and KRAS- mutated ctDNA at D0, as well as the presence of KRAS- mutated ctDNA at D28, were strongly associated with lower centralized disease control rate (cDCR), shorter cPFS and OS in multivariate analysis. A score combining cfDNA level at diagnosis ≥ or <30 ng/mL and presence or not of KRAS- mutated ctDNA at D28 was an optimal predictor of cDCR (OR=30.7, IC95% 4.31–218 P =.001), PFS (HR=6.79, IC95% 2.76–16.7, P <.001) and OS (HR=9.98, IC95% 4.14–24.1, P <.001). A combined score using cfDNA level at diagnosis and KRAS -mutated ctDNA at D28 is strongly associated with patient survival/response to chemotherapy in UPA. ClinicalTrials.gov Identifier: NCT04560270 [ABSTRACT FROM AUTHOR]

The purpose of the study was to summarize and analyze modern data about non-invasive methods of molecular diagnosis and approaches to the personalized therapy of diffuse midline glioma (DMG). Material and Methods. The search and analysis of publications was carried out using Google Scholar, Pubmed, Elsevier, Web of Science, Elibrary systems. The review includes publications published from 2011 to 2022. Of the 102 articles found, 59 were used to write the review. Results. In this review, we discuss the spectrum of somatic driver mutations present in DMG tumor cells and their relationship with the sensitivity of tumor cells to certain types of therapy - a pharmacogenetic approach to the selection of individual treatments (targeted therapy). We provide examples of new methods of targeted therapy for DMG, which are currently at the stage of preclinical laboratory development. Also, we discuss examples of the use of 3D cell cultures for the development of targeted therapies, including the use of perfusion systems. The review describes the methods of analysis of liquid biopsy, which allow the detection of tumor-specific biomarkers in the non-invasive diagnosis of DMG, including a number of methods that have not yet been tested in the clinic. The following is a list of tumor-specific biomarkers for diagnosing, monitoring, and selecting targeted therapy for DMG. Finally, we discuss the possibility of implementing these methods in the clinic and present the results of several clinical trials. Conclusion. In oncology, new methods of molecular genetics, such as analysis of liquid biopsy, allow diagnosis and monitoring of treatment in cases where classical methods that require tissue sampling are not applicable (for example, the analysis of genetically heterogeneous tumors and tumors of surgically inaccessible localization). These tumors include DMG, a primary brain tumor most common in children. The available data confirm the relevance of the search for new specific tumor biomarkers, as well as targets for targeted therapy of the paediatric-type diffuse gliomas.

In the precision medicine era of cystic fibrosis (CF), therapeutic interventions, by the so-called modulators, target the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The levels of targetable CFTR proteins are a main variable in the success of patient-specific therapy. In turn, the CFTR protein level depends, at least in part, on the level of CFTR mRNA. Many mechanisms can modulate the CFTR mRNA level, for example, transcriptional rate, stability of the mRNA, epigenetics, and pathogenic variants that can affect mRNA production and degradation. Independently from the causes of variable CFTR mRNA levels, their exact quantitative assessment is of great importance in CF. Methods with high analytical sensitivity, precision, and accuracy are mandatory for the quantitative evaluation aimed at the amelioration of the diagnostic, prognostic, and therapeutic aspects. This paper compares, for the first time, two CFTR gene expression quantification methods: a well-established method for the relative quantification of CFTR mRNA using a real-time PCR and an innovative method for its absolute quantification using a droplet digital PCR. No comprehensive methods for absolute CFTR quantification via droplet digital PCR have been published so far. The accurate quantification of CFTR expression at the mRNA level is a critical step for the personalized therapeutic approaches of CF. [ABSTRACT FROM AUTHOR]

Despite the success of current therapy concepts, patients with advanced non-small-cell lung cancer (NSCLC) still have a very poor prognosis. Therefore, biological markers are urgently needed, which allow the assessment of prognosis, or prediction of the success of therapy or resistance in this disease. Circulating microRNAs (miRs) have potential as biomarkers for the prognosis and prediction of response to therapy in cancer patients. Based on recent evidence that circulating miR-16, miR-29a, miR-144 and miR-150 can be regulated by ionizing radiation, the concentration of these four miRs was assessed in the plasma of NSCLC patients at different time points of radiotherapy by digital droplet PCR (ddPCR). Furthermore, their impact on patients' prognosis was evaluated. The mean plasma levels of miR-16, miR-29a, miR-144 and miR-150 significantly differed intra- and inter-individually, and during therapy in NSCLC patients, but showed a strong positive correlation. The individual plasma levels of miR-16, miR-29a and miR-144 had prognostic value in NSCLC patients during or at the end of radiotherapy in Cox's regression models. NSCLC patients with low levels of these three miRs at the end of radiotherapy had the worst prognosis. However, miR-150 plasma levels and treatment-dependent changes were not predictive. In conclusion, circulating miR-16, miR-29a and miR-144, but not miR-150, have a prognostic value in NSCLC patients undergoing radiotherapy. [ABSTRACT FROM AUTHOR]

Mycoplasma pneumoniae is a common causative pathogen of communityacquired pneumonia. An accurate and sensitive detection method is important for evaluating disease severity and treatment efficacy. Digital droplet PCR (ddPCR) is a competent method enabling the absolute quantification of DNA copy number with high precision and sensitivity. We established ddPCR for M. pneumoniae detection, using clinical specimens for validation, and this showed excellent specificity for M. pneumoniae. The limit of detection of ddPCR was 2.9 copies/reaction, while that for real-time PCR was 10.8 copies/reaction. In total, 178 clinical samples were used to evaluate the ddPCR assay, which correctly identified and differentiated 80 positive samples, whereas the real-time PCR tested 79 samples as positive. One sample that tested negative in real-time PCR was positive in ddPCR, with a bacterial load of three copies/test. For samples that tested positive in both methods, the cycle threshold of real-time PCR was highly correlated with the copy number of ddPCR. Bacterial loads in patients with severe M. pneumoniae pneumonia were significantly higher than those in patients with general M. pneumoniae pneumonia. The ddPCR showed that bacterial loads were significantly decreased after macrolide treatment, which could have reflected the treatment efficacy. The proposed ddPCR assay was sensitive and specific for the detection of M. pneumoniae. Quantitative monitoring of bacterial load in clinical samples could help clinicians to evaluate treatment efficacy. [ABSTRACT FROM AUTHOR]

Cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are significant complications in patients with relapsed/refractory diffuse large B-cell lymphoma undergoing chimeric antigen receptor T-cell (CAR-T cell) therapy. However, it remains unclear whether CAR-T cell expression itself is clinically relevant. We assessed CAR-T cell mRNA expression and DNA concentration by digital droplet PCR in peripheral blood from 14 sequential CAR-T cell recipients. Patients were grouped according to CAR-T cell peak expression. Patients with high CAR-T cell peak expression (8 patients; 57%) had higher rates of ICANS (p=0.0308) and intensive care unit admission (p=0.0404), longer durations of hospitalization (p=0.0077), and, although not statistically significant, a higher rate of CRS (p=0.0778). There was a correlation of CAR-T cell mRNA expression with DNA concentration, but CAR-T cell expression levels failed to correlate to response or survival. Our data suggest that higher CAR-T cell peak mRNA expression is associated with increased risk for ICANS and possibly CRS, requiring further investigation in larger studies. [ABSTRACT FROM AUTHOR]

Langerhans cell histiocytosis-associated neurodegenerative disease (LCH-ND) is the most serious late complication secondary to LCH and is gradually progressive, destructive, and irreversible. Detection of the BRAF V600E mutation in peripheral blood mononuclear cells (PBMCs), even in the absence of active LCH lesions, is considered a sign of clinical LCH-ND, presenting with both abnormal imaging findings and neurological symptoms. However, the detection of the BRAF V600E mutation in PBMCs of patients with asymptomatic radiological LCH-ND (rLCH-ND) without active LCH lesions who present only with abnormal imaging findings is unknown. In this study, we analyzed the BRAF V600E mutations in PBMCs and cell-free DNA (cfDNA) of patients with rLCH-ND without active LCH lesions (n = 5) using a droplet digital polymerase chain reaction (ddPCR) assay. The BRAF V600E mutation in PBMCs was detected in three out of five (60%) cases. The mutant allele frequencies in the three positive cases were 0.049%, 0.027%, and 0.015%, respectively. However, the cfDNA BRAF V600E mutation remained undetected in all patients. Detection of the BRAF V600E mutant allele in PBMCs may be helpful in identifying asymptomatic rLCH-ND in patients at high risk for developing LCH-ND, including those with relapses at CNS risk sites or central diabetes insipidus. [ABSTRACT FROM AUTHOR]

Minimal/measurable residual disease (MRD) monitoring is progressively changing the management of hematologic malignancies. The possibility of detecting the persistence/reappearance of disease in patients in apparent clinical remission offers a refined risk stratification and a treatment decision making tool. Several molecular techniques are employed to monitor MRD, from conventional realtime quantitative polymerase chain reaction (RQ-PCR) to next generation sequencing and digital droplet PCR (ddPCR), in different tissues or compartments through the detection of fusion genes, immunoglobulin and Tcell receptor gene rearrangements or disease-specific mutations. RQ-PCR is still the gold standard for MRD analysis despite some limitations. ddPCR, considered the third-generation PCR, yields a direct, absolute, and accurate detection and quantification of low-abundance nucleic acids. In the setting of MRD monitoring it carries the major advantage of not requiring a reference standard curve built with the diagnostic sample dilution and of allowing to reduce the number of samples below the quantitative range. At present, the broad use of ddPCR to monitor MRD in the clinical practice is limited by the lack of international guidelines. Its application within clinical trials is nonetheless progressively growing both in acute lymphoblastic leukemia as well as in chronic lymphocytic leukemia and non-Hodgkin lymphomas. The aim of this review is to summarize the accumulating data on the use of ddPCR for MRD monitoring in chronic lymphoid malignancies and to highlight how this new technique is likely to enter into the clinical practice. [ABSTRACT FROM AUTHOR]

Liquid biopsy for CNS tumors is in its nascent phase, hindered by the low levels of circulating tumor DNA (ctDNA). Overcoming this challenge requires highly sensitive molecular techniques. DD-PCR emerges as a standout technique due to its ability to identify rare mutations, copy number variations, and circulating nucleic acids, making it one of the best methods for identifying somatic mutations in cell-free DNA (cfDNA). Despite promising results from various studies demonstrating the feasibility of obtaining informative ctDNA profiles from liquid biopsy samples, challenges persist, including the need to standardize sample collection, storage, and processing methods, define clear assay positivity thresholds, and address the overall low assay sensitivity. Our two-phase study began by assessing DD-PCR efficacy in FFPE tissues, revealing robust concordance with immunohistochemistry. In Phase 1 (85 cases), DD-PCR on FFPE tissues demonstrated 100 % sensitivity and specificity for IDH1 R132H mutations. In Phase 2 (100 cases), analysis extended to cfDNA, maintaining high specificity (100 %) with moderate sensitivity (44.2 %). Overall concordance with immunohistochemistry was 61 %, highlighting liquid biopsy's potential in glioma management. The findings emphasized DD-PCR's clinical utility in both tissue and liquid biopsy, underscoring its role in early detection, diagnosis, and therapeutic monitoring of diffuse gliomas. • Detection of IDH1 R132H Mutation: The study successfully detected the IDH1 R132H mutation in the plasma of glioma patients using a highly sensitive droplet digital PCR (DD-PCR) assay. • Comparison with Sanger Sequencing : DD-PCR outperformed Sanger sequencing in detecting mutations, with a 100 % concordance with immunohistochemistry (IHC), compared to 95.4 % with Sanger sequencing. • Clinical Utility of Liquid Biopsy: Demonstrated the potential of liquid biopsy for glioma, achieving a sensitivity of 44 % at 100 % specificity in detecting IDH1 R132H mutations in plasma cfDNA samples. • Addressing Challenges: Highlighted issues like tumor heterogeneity and blood-brain barrier limitations, which can impact plasma mutation detection, emphasizing the need for standardized methods and larger studies for clinical application. • Pioneering Work in India: This is reported as the first study in India to successfully detect IDH1 R132H mutations from plasma liquid biopsy samples in glioma patients highlighting the promise of liquid biopsy techniques in resource-limited settings. [ABSTRACT FROM AUTHOR]

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient–dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection.

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. HIGHLIGHTS RT-digital droplet PCR was used to detect B.1.1.7 SARS-CoV-2 variant in sewage.; Signature mutations N501Y and del H69/V70 were detected in equal concentrations.; Signature mutations of B.1.1.7 became dominant within 6 weeks in Hungarian wastewater treatment plants.; Sewage data closely correlated with clinical emergence of B.1.1.7 cases.; Wastewater-based epidemiology is an efficient tool in tracing emerging SARS-CoV-2 variants of concern.;

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination.

Minimal/measurable residual disease (MRD) monitoring is progressively changing the management of hematologic malignancies. The possibility of detecting the persistence/reappearance of disease in patients in apparent clinical remission offers a refined risk stratification and a treatment decision making tool. Several molecular techniques are employed to monitor MRD, from conventional real-time quantitative polymerase chain reaction (RQ-PCR) to next generation sequencing and digital droplet PCR (ddPCR), in different tissues or compartments through the detection of fusion genes, immunoglobulin and T-cell receptor gene rearrangements or disease-specific mutations. RQ-PCR is still the gold standard for MRD analysis despite some limitations. ddPCR, considered the third-generation PCR, yields a direct, absolute, and accurate detection and quantification of low-abundance nucleic acids. In the setting of MRD monitoring it carries the major advantage of not requiring a reference standard curve built with the diagnostic sample dilution and of allowing to reduce the number of samples below the quantitative range. At present, the broad use of ddPCR to monitor MRD in the clinical practice is limited by the lack of international guidelines. Its application within clinical trials is nonetheless progressively growing both in acute lymphoblastic leukemia as well as in chronic lymphocytic leukemia and non-Hodgkin lymphomas. The aim of this review is to summarize the accumulating data on the use of ddPCR for MRD monitoring in chronic lymphoid malignancies and to highlight how this new technique is likely to enter into the clinical practice.

ABSTRACT Accurate and timely etiological diagnosis is crucial for bloodstream infections (BSIs) due to their high disability and mortality. We conducted a single-center prospective cohort study to compare the digital droplet PCR (ddPCR) assay with traditional blood culture. A total of 169 blood samples from 122 patients with suspected BSIs were collected, mostly from the department of infectious diseases, the emergency department, and the intensive care units, and the clinical data were also recorded. Nucleic acid was extracted from the blood samples, and a 5-fluorescent-channel droplet digital PCR assay was performed and then fed back with the pathogen and its copies. In BSI patients, ddPCR reported an overall 85.71% (12/14) (95% confidence interval [CI], 56.15 to 97.48%) sensitivity, 100% (7/7) (95% CI, 56.09 to 100.00%) and 71.43% (5/7) (95% CI, 30.26 to 94.89%) sensitivity in patients without empirical treatment and in empirically treated patients, respectively. Compared to traditional blood culture, the overall detection rate of ddPCR was significantly higher, 11.27% (16/142) (95% CI, 6.78 to 17.93%) versus 30.28% (43/142) (95% CI, 23.01 to 38.64%), and the extra detection rate of ddPCR was 19.01% (27/142) (95% CI, 13.11 to 26.63%). Of the ddPCR-positive culture-negative cases, 74.19% (23/31) (95% CI, 55.07 to 87.46%) were consistent with the final clinical diagnosis, including 10 bacteria and fungi. The detection rate of ddPCR was significantly higher in patients with white blood cell (WBC) counts of >10 · 109/L, C-reactive protein (CRP) of >70 mg/L, or procalcitonin (PCT) of >0.9 ng/L. Pathogen loads detected by ddPCR are correlated with WBC, CRP, and especially, PCT levels, precisely and rapidly reflecting clinical disease progression. ddPCR has an important guiding value for the clinical use of antibiotics to achieve the best pathogen coverage and the antibacterial effect. Collectively, ddPCR showed a great diagnostic performance in BSIs and had an overall higher detection rate than blood culture. In addition, ddPCR could be used to dynamically monitor the disease progression and provide medication guidance on antibiotic use. IMPORTANCE ddPCR is a promising method to address the current challenges of BSI diagnosis and precise treatment, as it is highly efficient in DNA detection. It shortens the identification of BSI-related pathogens from several days of traditional bacterial culture to 4 to 5 h. It is extremely sensitive and more tolerant to PCR inhibitors, which may facilitate the amplification and enable the detection of a meager amount of DNA fragments in detecting BSI-related pathogens and drug-resistant genes. It can identify almost 20 pathogens in one reaction, which reduces the usage of clinical blood samples to no more than 2 mL. Additionally, dynamic monitoring, assessment of pathogens, and antibiotic resistance genes in patients could be used to guide timely and precise adjustment of antimicrobial prescription. The short turnaround time of ddPCR may have the potential to guide antimicrobial treatment in the very early stage of sepsis and reduce the mortality and disability rate of sepsis.

Introduction: Extracellular vesicles (EVs) have emerged as a very attractive source of cancer- derived RNA biomarkers for the early detection, prognosis and monitoring of various cancers, including prostate cancer (PC). However, biofluids contain a mixture of EVs released from a variety of tissues and the fraction of total EVs that are derived from PC tissue is not known. Moreover, the optimal biofluid—plasma or urine—that is more suitable for the detection of EV- enclosed RNA biomarkers is not yet clear.Methodology: In the current study, we performed RNA sequencing analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer.Results and Discussion: The most abundant RNA biotypes in EVs were miRNA, piRNA, tRNA, lncRNA, rRNA and mRNA. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype. The levels of 63 mRNAs, 3 lncRNAs, 2 miRNAs and 1 piRNA were significantly increased in the tumors and decreased in the post-operation urinary EVs, thus suggesting that these RNAs mainly originate from PC tissue. No such RNA biomarkers were identified in plasma EVs. This suggests that the fraction of PC-derived EVs in urine is larger than in plasma and allows the detection and tracking of PC-derived RNAs.

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. [ABSTRACT FROM AUTHOR]

Keywords: digital droplet PCR; Midichloria-like organism; rainbow trout; recirculating aquaculture system; red mark syndrome EN digital droplet PCR Midichloria-like organism rainbow trout recirculating aquaculture system red mark syndrome 1889 1892 4 11/11/22 20221201 NES 221201 Red mark syndrome is a non-lethal skin disease, reported to affect farmed rainbow trout over 50 g worldwide (Metselaar et al., 2022). The manuscript by Oh et al. (2019) describes several cases of the disease in rainbow trout in South Korea, in which macroscopic lesions resembling RMS can be seen in juvenile rainbow trout weighed less than 30 g. [Extracted from the article]

Multiparameter flow cytometry (MFC)-based minimal residual disease has been a poor predictor of prognosis in children with acute myeloid leukemia (AML). This study aimed to evaluate the incremental value of serial monitoring by droplet digital PCR (ddPCR) in forecasting the outcome of AML. Twenty-four children with AML were enrolled and the relapse-free survival (RFS) rate was estimated using the Kaplan-Meier method. Survival estimates were compared using the log-rank test. Survival analysis showed that the RFS rate in the ddPCR ≥ 0.1% group was significantly lower than that in the < 0.1% group (35.7% ± 19.8% vs. 83.6% ± 10.8%, P = 0.003). Moreover, serial monitoring by ddPCR showed that some mutations remained positive in some patients even though other co-mutations were eliminated, and those patients were more prone to relapse, with a significantly poorer RFS compared to patients negative for mutation (22.0% ± 19.2% vs 83.3% ± 11.3%, P = 0.001). Consequently, ddPCR may assist in prognostic forecasting for pediatric AML. [ABSTRACT FROM AUTHOR]

Simple Summary: Ruminants produce methane, a potent greenhouse gas, as a byproduct of microbial activity during ruminal fermentation. To lessen greenhouse gases emissions, it is necessary to evaluate methane-reducing feeding alternatives for ruminants, such as the use of tropical trees and shrubs. The secondary metabolites produced by these plants have been shown to reduce ruminal methane emissions. Here, we evaluated the effect of the foliage of eleven tropical trees and shrubs edible for cattle in ruminal fermentation, methane production and microbiota in an in vitro assay. The content of condensed tannins and saponins in the foliage was variable between the species of plants. We observed a reduction in methane production and changes in microbial populations depending on the species of tree or shrub. Additionally, condensed tannins reduced methane production. The inclusion of the foliage of tropical trees and shrubs in the diet of ruminants may be a step towards diminishing greenhouses gases emissions, and therefore reducing the contribution to global warming from cattle production. Ruminants, mainly cattle, contribute to greenhouse gases (GHG) emissions as methane (CH4) is produced by ruminal fermentation. Hence, various anti-methanogenic feed strategies have been studied, including the use of plants with secondary metabolites. This study evaluated in vitro ruminal fermentation metrics, microbial composition by digital droplet PCR (ddPCR) and the CH4 production of the foliage of several tropical trees and shrubs: Leucaena leucocephala, Moringa oleifera, Albizia lebbeck, Enterolobium cyclocarpum, Piscidia piscipula, Brosimum alicastrum, Lysiloma latisiliquum, Guazuma ulmifolia, Cnidoscolus aconitifolius, Gliricidia sepium and Bursera simaruba, using Cynodon plectostachyus grass as control. The results showed a wide variation in the chemical composition of the foliage, as well as in the ruminal microbiota. The crude protein (CP) content ranged from 11 to 25%, whereas the content of condensed tannins (CT) and saponins (S) was from 0.02 to 7%, and 3.2 to 6.6%, respectively. The greatest dry matter degradability (DMD) after 72 h was 69% and the least 35%, the latter coinciding with the least gas production (GP). A negative correlation was found between the CT and CH4 production, also between protozoa and fungi with the SGMT group of archaea. We concluded that the foliage of some tropical trees and shrubs has a high nutritional value and the potential to decrease CH4 production due to its CT content. [ABSTRACT FROM AUTHOR]

Objective: Chimerism analysis is an important post-transplant assessment for allogeneic hematopoietic stem cell transplant (HCT) recipients. Although various chimerism analysis techniques are already established, they are limited in terms of sensitivity, versatility, and turnaround time. Our objective was to develop a digital droplet polymerase chain reaction (ddPCR) assay for chimerism analysis using ABO gene polymorphisms as markers., Methods: Our new chimerism analysis method utilizes ddPCR to assess the ABO gene polymorphisms that encode the ABO blood genotype. ABO genotypes were determined in blood samples from 15 HCT recipients using the O panel (rs8176719) and B panel (rs8176746 and rs8176747)., Results: The two panels distinguished six ABO genotypes (AA, AO, BB, BO, AB, and OO). The results of chimerism analysis using ABO genotypes with ddPCR were compatible with those of established methods, such as SRY gene analysis and the use of short tandem repeat markers via standard PCR. Our method could distinguish chimerism in 77% of donor and recipient combinations in the Japanese population., Conclusions: We developed a sensitive and rapid chimerism analysis method for HCT using ABO gene polymorphisms in ddPCR., Competing Interests: Declarations. Conflict of interest: There are no conflicts of interest to declare., (© 2024. Japanese Society of Hematology.)

Background: Relapse remains the primary cause of death after hematopoietic cell transplantation (HCT) for acute leukemia. The ability to identify minimal/measurable residual disease (MRD) via the blood could identify patients earlier when immunologic interventions may be more successful. We evaluated a new test that could quantify blood tumor mRNA as leukemia MRD surveillance using droplet digital PCR (ddPCR). Methods: The multiplex ddPCR assay was developed using tumor cell lines positive for the tumor associated antigens (TAA: WT1, PRAME, BIRC5), with homeostatic ABL1. On IRB-approved protocols, RNA was isolated from mononuclear cells from acute leukemia patients after HCT (n = 31 subjects; n = 91 specimens) and healthy donors (n = 20). ddPCR simultaneously quantitated mRNA expression of WT1, PRAME, BIRC5, and ABL1 and the TAA/ABL1 blood ratio was measured in patients with and without active leukemia after HCT. Results: Tumor cell lines confirmed quantitation of TAAs. In patients with active acute leukemia after HCT (MRD+ or relapse; n=19), the blood levels of WT1/ABL1, PRAME/ABL1, and BIRC5/ABL1 exceeded healthy donors (p<0.0001, p=0.0286, and p=0.0064 respectively). Active disease status was associated with TAA positivity (1+ TAA vs 0 TAA) with an odds ratio=10.67, (p=0.0070, 95% confidence interval 1.91 – 59.62). The area under the curve is 0.7544. Changes in ddPCR correlated with disease response captured on standard of care tests, accurately denoting positive or negative disease burden in 15/16 (95%). Of patients with MRD+ or relapsed leukemia after HCT, 84% were positive for at least one TAA/ABL1 in the peripheral blood. In summary, we have developed a new method for blood MRD monitoring of leukemia after HCT and present preliminary data that the TAA/ABL1 ratio may may serve as a novel surrogate biomarker for relapse of acute leukemia after HCT. [ABSTRACT FROM AUTHOR]

Circulating cell-free HPV DNA (ccfHPV DNA) may serve as a marker for cervical cancer. In this study, we used digital droplet PCR (ddPCR) to detect and quantify ccfHPV DNA in plasma from patients with HPV16- or HPV18-associated cervical cancer. Blood samples from 60 patients diagnosed with cervical cancer (FIGO IA1-IVA) at Aarhus or Odense University Hospital (June 2018 to March 2020) were collected prior to treatment, and patients were subdivided into an early stage (n = 30) and a late-stage subgroup (n = 30) according to disease stage. Furthermore, blood samples from eight women with HPV16- or 18-associated premalignant conditions (CIN3), and 15 healthy controls were collected. ddPCR was used to analyze plasma from all participants. ccfHPV DNA was detected in 19 late-stage patients (63.33%), 3 early stage patients (10.00%), and none of the CIN3 patients or controls. Quantitative evaluation showed significant correlations between ccfHPV DNA level and stage, tumor score, and tumor size. Thus, our results indicate that ccfHPV DNA may not be a useful marker for early detection of cervical cancer. However, for patients with advanced stage cervical cancer, ccfHPV DNA level represents a promising tool to establish tumor burden, making it useful for establishing treatment response and monitoring the disease. [ABSTRACT FROM AUTHOR]

BackgroundRelapse remains the primary cause of death after hematopoietic cell transplantation (HCT) for acute leukemia. The ability to identify minimal/measurable residual disease (MRD) via the blood could identify patients earlier when immunologic interventions may be more successful. We evaluated a new test that could quantify blood tumor mRNA as leukemia MRD surveillance using droplet digital PCR (ddPCR).MethodsThe multiplex ddPCR assay was developed using tumor cell lines positive for the tumor associated antigens (TAA: WT1, PRAME, BIRC5), with homeostatic ABL1. On IRB-approved protocols, RNA was isolated from mononuclear cells from acute leukemia patients after HCT (n = 31 subjects; n = 91 specimens) and healthy donors (n = 20). ddPCR simultaneously quantitated mRNA expression of WT1, PRAME, BIRC5, and ABL1 and the TAA/ABL1 blood ratio was measured in patients with and without active leukemia after HCT.ResultsTumor cell lines confirmed quantitation of TAAs. In patients with active acute leukemia after HCT (MRD+ or relapse; n=19), the blood levels of WT1/ABL1, PRAME/ABL1, and BIRC5/ABL1 exceeded healthy donors (p

Lifelong treatment of pediatric chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs) can affect their growth and development. For these reasons, clinical trials have explored the feasibility of TKI discontinuation in children with a sufficient TKI response. We evaluated the analytical performance of digital droplet PCR (ddPCR) to quantify BCR-ABL1 and compared the results with reverse transcription quantitative polymerase chain reaction (RT-qPCR). We further investigated whether ddPCR could be used to determine TKI discontinuation in a clinical setting. Performance of ddPCR was evaluated using standard materials for BCR-ABL1, and a total of 197 clinical samples from 45 pediatric CML patients was included for comparison with RT-qPCR. ddPCR showed excellent analytical sensitivity with 0.001% international scale (IS) and linearity with R2 > 0.99 in log scale. BCR-ABL1 % IS results correlated well with those of RT-qPCR (R2 = 0.9435), however, they showed a moderate strength for agreement with a Cohen's kappa of 0.41 due to higher sensitivity of ddPCR. Among 45 pediatric CML patients, 42 were treated with first-line TKIs including imatinib (n = 27, 64%) and dasatinib (n = 12, 29%), and three patients that were started with imatinib were switched to dasatinib. When we evaluated whether follow-up samples fulfilled ABL1 copies ≥ 10,000 required for deep molecular response (DMR), all samples were acceptable by ddPCR, whereas 18% by RT-qPCR did not reached acceptable ABL1 copies. Moreover, 52 and 13% reached ABL1 copies ≥ 32,000 required for MR4.5 by ddPCR and RT-qPCR, respectively. Seven patients discontinued TKI and the median TKI treatment duration was 73 months prior to discontinuation. Prior to discontinuation, the median duration of sustained undetected BCR-ABL1 was 60 months. Two patients experienced loss of major MR (MMR) during follow-up and restarted dasatinib 5 months after discontinuation. They achieved MMR again and maintained better than DMR afterward. Results from those patients demonstrated that RT-qPCR did not match the need for adequate ABL1 copies for MR4.5 while majority of ddPCR could. Therefore, ddPCR was technically more acceptable to decide and monitor pediatric CML patients before and after TKI discontinuation.

Abstract The application of eDNA techniques for the detection, monitoring, and conservation of biodiversity holds great promise. While many studies apply eDNA techniques in aquatic systems to determine the presence or absence of a given species, using eDNA for the purpose of species density or biomass predictions remains a challenge, especially for freshwater invertebrates that shed exoskeletons. Here, we aimed to determine whether and how eDNA concentrations relate to exoskeleton‐shedding invertebrate densities. We used microcosms holding different densities of a common invertebrate freshwater species, Daphnia magna. During 2 weeks, we monitored temporal dynamics of eDNA and the eDNA/density relationship by taking water samples and quantifying eDNA concentrations with the droplet digital PCR. The setup included one treatment without and one with homogenization before sampling, to test the effects of admixture on the relation between eDNA concentration and density. Daphnia magna individuals were removed after 1.5 weeks to track DNA degradation rates. In the stagnant water setup, hardly any DNA was detected before D. magna removal. Within days after removal, eDNA concentrations became undetectable. No significant correlation between D. magna density and eDNA concentrations was observed. In the homogenization treatment, a significant positive correlation between eDNA concentration and densities was demonstrated for the days around D. magna removal, albeit with some within‐treatment variability. Our results show that, given adequate time for eDNA production and degradation to stabilize, positive correlations between eDNA and organism densities in water with sufficient homogenization are detectable for exoskeleton‐shedding invertebrates. Therefore, our study indicates that—although difficult—using eDNA to quantify freshwater exoskeleton‐shedding invertebrate densities may be possible under field conditions if circumstances result in frequent homogenization of the water column.

Enteropathies are a serious concern in racing pigeons as they significantly impair performance in races and their training, and viruses are suspected to be one of the main factors. Astroviruses are well-known to be responsible for causing enteric disease in humans and various other animals including birds, although their prevalence and pathogenicity in pigeons is poorly understood. In this study, we investigated 2 groups of young racing pigeons (sick—study group and healthy—control group) to assess the correlation between the number of astrovirus genome copies in cloacal swabs and the occurrence of enteropathy. To determine this, we developed a novel TaqMan quantitative PCR (qPCR) and digital droplet PCR (ddPCR) methods for astrovirus detection and absolute quantitative analysis. We also performed high-throughput sequencing to obtain the complete genome sequences and establish the genetic similarity of the obtained strains to known astroviruses of poultry and other avian species. Two new complete genome sequences of pigeon astroviruses in the Avastrovirus genus were identified, representing 2 new species. These were found most closely related to astroviruses identified in Columbidae species and chickens. They share an average of 75.8% genome-wide pairwise identity and 57.6% and 64.6% capsid protein sequence identity with other unclassified columbid avastrovirus sequences in GenBank. Although the difference in prevalence of astrovirus in the study and control group was found statistically insignificant, there was a significant difference between the number of genome copies in positive samples from both groups. These unambiguous results leave the role of astroviruses as enteropathogenic factors in pigeons still undetermined. [ABSTRACT FROM AUTHOR]

Penile carcinoma develops either through human papillomavirus (HPV) related or unrelated carcinogenic pathways. Genetic alterations and nucleotide changes in coding regions (ie, TP53, CDKN2A, PIK3CA and NOTCH1) are main cancer driver events either in HPV positive or in HPV negative tumours. We investigated the presence of hotspot nucleotide mutations in TERT promoter (TERTp) and PIK3CA exon 9 and their relationship with HPV status in 69 penile cancer cases from Italian and Ugandan patients. Genetic variations and viral sequences have been characterised by end‐point polymerase chain reaction (PCR) and Sanger sequencing. The mutant allele frequencies (MAFs) of TERTp −124A/−146A and PIK3CA E545K have been determined by droplet digital PCR (ddPCR) assays. The results showed that TERTp mutations are highly prevalent in penile carcinoma (53.6%) and significantly more frequent in HPV negative (67.6%) than HPV positive (32.4%) cases (P =.0482). PIK3CA mutations were similarly distributed in virus‐related and unrelated cases (25.9% and 26.7%, respectively) and coexisted with TERTp changes in 15.8% of penile carcinoma samples. Notably, MAFs of co‐occurring mutations were frequently discordant indicating that PIK3CA E545K nucleotide changes are subsequent genetic events occurring in subclones of TERTp mutated cells. The frequencies of TERTp and PIK3CA mutations were higher among Italian compared to Ugandan cases and inversely correlated with the HPV status. In conclusion, TERTp mutations are very common in penile carcinoma and their coexistence with PIK3CA in a substantial number of cases may represent a novel oncogenic synergy relevant for patient stratification and use of therapeutic strategies against new actionable targets. [ABSTRACT FROM AUTHOR]

Abstract Background It is still unclear whether epidermal growth factor receptor (EGFR) mutation of primary lung adenocarcinoma can be detected on sputum samples. This study aimed to examine EGFR mutations of primary lung adenocarcinoma in sputum samples using droplet digital polymerase chain reaction (ddPCR) and compare it with an EGFR mutation in surgically resected lung cancer. Methods Sputum was prospectively collected from the patients before complete resection of the primary lung cancer at Kanagawa Cancer Center from September 2014 to May 2016. ddPCR was performed to detect EGFR exon 21 L858R point mutation (Ex21) and EGFR exon 19 deletion mutation (Ex19) in sputum samples from patients with lung adenocarcinoma. The concordance of EGFR mutation status in sputum samples and tumors in surgically resected specimen was evaluated for each positive and negative cytology group. Results One hundred and eighteen patients with primary lung adenocarcinoma provided sputum samples. Sputum cytology was positive in 13 patients (11.0%). ddPCR detected two cases of Ex21 and two cases of Ex19 in sputum cytology positive cases. Compared to surgically resected specimens, the sensitivity, specificity, and positive predictive value of EGFR mutation (Ex19 and Ex21) detection were 80.0%, 100%, and 100%, respectively, in sputum cytology positive cases. In contrast, the sensitivity, specificity, and positive predictive value of EGFR mutation (Ex19 and Ex21) detection were 3.1%, 100%, and 100%, respectively, in sputum cytology negative cases. Conclusions EGFR mutations in primary lung adenocarcinoma can be detected with high sensitivity in sputum samples if sputum cytology is positive.

Quantification of tumor‐specific variants (TSVs) in cell‐free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next‐generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell‐free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell‐free DNA to its full potential.

Low-grade serous ovarian cancer (LGSOC) poses a specific clinical challenge due to advanced presentation at diagnosis and the lack of effective systemic treatments. The aim of this study was to use a precision medicine approach to identify clinically actionable mutations in a patient with recurrent LGSOC. Primary, metastatic and recurrence tissue, and blood samples were collected from a stage IV LGSOC patient. Single-gene testing for clinically actionable mutations (BRAF V600, KRAS and NRAS) and subsequent whole-exome sequencing (WES) were performed. Droplet digital PCR was used to evaluate the presence of an identified BRAF D594G mutation in the matched plasma cell-free DNA (cfDNA). No clinically actionable mutations were identified using single-gene testing. WES identified a BRAF D594G mutation in six of seven tumor samples. The patient was commenced on a MEK inhibitor, trametinib, but with minimal clinical response. A newly designed ddPCR assay detected the BRAF alteration in the matched tissues and liquid biopsy cfDNA. The identification and sensitive plasma detection of a common “druggable” target emphasises the impact of precision medicine on the management of rare tumors and its potential contribution to novel monitoring regimens in this field.

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 ¼ 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. [ABSTRACT FROM AUTHOR]

Background: Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict posttransplant immune‐related complications, and eventually to customize immunosuppression. Methods: In this study, 327 blood samples were tested using two real‐time PCR (rtPCR) assays both targeted to the untranslated region of the TTV genome. The first assay was an in‐house rtPCR developed by our group, the second one was the recently marketed TTV R‐GENE assay. Results: In the validation study, the TTV R‐GENE showed good performances in precision and reproducibility, and sensitivity as low as 12 TTV DNA copies/mL, like previously reported for the in‐house rtPCR. The Bland‐Altman analysis showed that the mean difference between the two methods was −0.3 log copies/mL. In the comparison study, 69% and 72% of samples were detected positive by rtPCR and TTV R‐GENE, respectively (94% concordance, κ = 0.88). Performances did not differ between the two rtPCRs by type of TTV group examined. When a newly‐developed in‐house digital droplet PCR was applied for TTV quantification and used as an alternative method of comparison on 94 samples, the results strongly correlated with those obtained by the two rtPCR methods (99% concordance). Conclusion: In summary, all the molecular methods assayed are highly sensitive and accurate in quantitation of TTV DNA in blood samples. Highlight: TTV monitoring is a promising strategy for quantification of the immunocompetence in transplant patients.PCR methods actually in use have comparable performances in TTV quantification [ABSTRACT FROM AUTHOR]

Abstract Development of resistance to platinum and poly(ADP‐ribose) polymerase inhibitors via secondary BRCA gene mutations that restore functional homologous recombination has been observed in a number of cancer types. Here we report a case of somatic BRCA2 mutation in a patient with high grade serous ovarian carcinoma. A secondary mutation predicted to restore the BRCA2 open reading frame was detected at low frequency (2.3%) in whole exome sequencing of a peritoneal biopsy at disease progression after treatment that included carboplatin and olaparib. We used digital droplet PCR (ddPCR) to verify the presence and frequency of this mutation in the biopsy sample at progression and also used this approach to assess the presence of the secondary mutation in preceding biopsies at diagnosis and first relapse. We found no evidence for the secondary mutation being present prior to the final progression biopsy, suggesting that this mutation was acquired late in the course of treatment. ddPCR provides a sensitive and specific technique to investigate the presence of low frequency mutations in a time series of biopsies.

The coronavirus disease-19 pandemic has resulted in a significant global health crisis, causing hundreds of millions of cases and millions of deaths. Despite being declared endemic, SARS-CoV-2 infection continues to pose a significant risk, particularly for immunocompromised individuals, highlighting the need for a more sensitive and specific detection. Reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) possesses a sensitive and absolute quantification compared to the gold standard. This study is the first to optimize RT-ddPCR for detecting SARS-CoV-2 in saliva specimens using a commercially available RT-qPCR kit. Optimization involved the assessment of the RT-ddPCR reaction mixture, annealing temperature adjustments, and validation using 40 stored saliva specimens. RT-qPCR was used as a reference method in this study. Compatibility assessment revealed that ddPCR Supermix for Probes (no dUTP) was preferable with an optimal annealing temperature of 57.6°C. Although a 25% higher primer/probe concentration provides a higher amplitude in droplet separation of positive control, the number of copy numbers decreased. An inverse correlation between Ct value and copy number concentration was displayed, presenting that the lower the Ct value, the higher the concentration, for the N and E genes with r 2 values of 0.98 and 0.85, respectively. However, ORF1ab was poorly correlated (r 2 of 0.34). The sensitivity of targeted and E genes was 100% and 93.3%, respectively; as for the specificity, the percentage ranged from 80.8% to 91.3%. This study implicates the applicability of a modified method in the ddPCR platform for similar types of pathogens using saliva specimens., (© The Author(s) 2024. Published by Oxford University Press.)

European brown hare (Lepus europaeus Pallas 1778) is a broadly distributed lagomorph species in Europe, recognized as a host for Ixodes ricinus and reservoir of a wide range of pathogens with zoonotic potential. Even though Lepus europaeus represents an important game animal in Central Europe, the data available on Anaplasma phagocytophilum in this lagomorph are scarce. In this study, three populations of brown hare from distinct localities in the Czech Republic were analysed for the presence of Anaplasma phagocytophilum DNA. We used standard qPCR, targeting the msp2 gene and adapted the same assay also for digital droplet PCR. Out of 91 samples, these two methods identified 9 and 12 as positive, respectively. For taxonomic analysis, we amplified the groEL gene from five of six samples that were found positive by both methods. In phylogenetic analyses, this haplotype belongs to ecotype 1, and to the subclade with isolates from cervids and I. ricinus. Our findings underline the importance of correct result interpretation and positivity cut‐off set‐up for different detection methods of A. phagocytophilum. This bacterium is characterized by a high intraspecific variability and highly sensitive detection itself, is not enough. Detailed molecular typing is necessary to define the zoonotic potential of different strains and their natural reservoirs. [ABSTRACT FROM AUTHOR]

Background: Early HIV diagnosis allows combination antiretroviral therapy (cART) initiation in the first days of life following in utero (IU) infection. The impact of early cART initiation on infant viral reservoir size in the setting of high-frequency cART nonadherence is unknown.Methods: Peripheral blood total HIV DNA from 164 early treated (day 0-21 of life) IU HIV-infected South African infants was measured using droplet digital PCR at birth and following suppressive cART. We evaluated the impact of cART initiation timing on HIV reservoir size and decay, and on the risk of subsequent plasma viremia in cART-suppressed infants.Results: Baseline HIV DNA (median 2.8 log10 copies/million peripheral blood mononuclear cells, range 0.7-4.8) did not correlate with age at cART initiation (0-21 days) but instead with maternal antenatal cART use. In 98 infants with plasma viral suppression on cART, HIV DNA half-life was 28 days. However, the probability of maintenance of plasma aviremia was low (0.46 at 12 months) and not influenced by HIV DNA load. Unexpectedly, longer time to viral suppression was associated with protection against subsequent viral rebound.Conclusions: With effective prophylaxis against mother-to-child transmission, cART initiation timing in the first 3 weeks of life is not critical to reservoir size. [ABSTRACT FROM AUTHOR]