Droplet Digital Polymerase Chain Reaction Research Articles

Amplification- and Enzyme-Free Magnetic Diagnostics Circuit for Whole-Genome Detection of SARS-CoV-2 RNA.

Polymerase chain reaction (PCR) requires thermal cycling and enzymatic reactions for sequence amplification, hampering their applications in point-of-care (POC) settings. Magnetic bioassays based on magnetic particle spectroscopy (MPS) and magnetic nanoparticles (MNPs) are isothermal, wash-free, and can be quantitative. Realizing them amplification- and enzyme-free on a benchtop device, they will become irreplaceable for POC applications. Here we demonstrate a first-in-class magnetic signal amplification circuit (MAC) that enables detection of whole genome of SARS-CoV-2 by combining the specificity of toehold-mediated DNA strand displacement with the magnetic response of MNPs to declustering processes. Using MAC, we detect the N gene of SARS-CoV-2 samples at a concentration of 104 RNA copies/µl as determined by droplet digital PCR. Further, we demonstrate that MAC can reliably distinguish between SARS-CoV-2 and other human coronaviruses. Being a wash-, amplification- and enzyme-free biosensing concept and working at isothermal conditions (25 °C) on a low-cost benchtop MPS device, our MAC biosensing concept offers several indispensable features for translating nucleic acid detection to POC applications.

Blood RNA Biomarkers Identify Bacterial and Biofilm Coinfections in COVID-19 Intensive Care Patients.

Purpose: Secondary opportunistic coinfections are a significant contributor to morbidity and mortality in intensive care unit (ICU) patients, but can be difficult to identify. Presently, new blood RNA biomarkers were tested in ICU patients to diagnose viral, bacterial, and biofilm coinfections. Methods: COVID-19 ICU patients had whole blood drawn in RNA preservative and stored at -80°C. Controls and subclinical infections were also studied. Droplet digital polymerase chain reaction (ddPCR) quantified 6 RNA biomarkers of host neutrophil activation to bacterial (DEFA1), biofilm (alkaline phosphatase [ALPL], IL8RB/CXCR2), and viral infections (IFI27, RSAD2). Viral titer in blood was measured by ddPCR for SARS-CoV2 (SCV2). Results: RNA biomarkers were elevated in ICU patients relative to controls. DEFA1 and ALPL RNA were significantly higher in severe versus incidental/moderate cases. SOFA score was correlated with white blood cell count (0.42), platelet count (-0.41), creatinine (0.38), and lactate dehydrogenase (0.31). ALPL RNA (0.59) showed the best correlation with SOFA score. IFI27 (0.52) and RSAD2 (0.38) were positively correlated with SCV2 viral titer. Overall, 57.8% of COVID-19 patients had a positive RNA biomarker for bacterial or biofilm infection. Conclusions: RNA biomarkers of host neutrophil activation indicate the presence of bacterial and biofilm coinfections in most COVID-19 patients. Recognizing coinfections may help to guide the treatment of ICU patients.

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

Identification of 'Candidatus Liberibacter asiaticus', the Huanglongbing Bacterium, in Citrus from Colombia.

'Candidatus Liberibacter' spp. are the most prevalent microorganisms in the citrus plant, associated with citrus huanglongbing, which are transmitted by psyllid vectors. In Colombia, the vector Diaphorina citri Kuwayama has been reported in different regions, but 'Ca. Liberibacter asiaticus' (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1/OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a/CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we used droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of the ribonucleotide reductase gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mitochondrial DNA and Electron Transport Chain Protein Levels Are Altered in Peripheral Nerve Tissues from Donors with HIV Sensory Neuropathy: A Pilot Study.

Distal sensory polyneuropathy (DSP) and distal neuropathic pain (DNP) remain significant challenges for older people with HIV (PWH), necessitating enhanced clinical attention. HIV and certain antiretroviral therapies (ARTs) can compromise mitochondrial function and impact mitochondrial DNA (mtDNA) replication, which is linked to DSP in ART-treated PWH. This study investigated mtDNA, mitochondrial fission and fusion proteins, and mitochondrial electron transport chain protein changes in the dorsal root ganglions (DRGs) and sural nerves (SuNs) of 11 autopsied PWH. In antemortem standardized assessments, six had no or one sign of DSP, while five exhibited two or more DSP signs. Digital droplet polymerase chain reaction was used to measure mtDNA quantity and the common deletions in isolated DNA. We found lower mtDNA copy numbers in DSP+ donors. SuNs exhibited a higher proportion of mtDNA common deletion than DRGs in both groups. Mitochondrial electron transport chain (ETC) proteins were altered in the DRGs of DSP+ compared to DSP- donors, particularly Complex I. These findings suggest that reduced mtDNA quantity and increased common deletion abundance may contribute to DSP in PWH, indicating diminished mitochondrial activity in the sensory neurons. Accumulated ETC proteins in the DRG imply impaired mitochondrial transport to the sensory neuron's distal portion. Identifying molecules to safeguard mitochondrial integrity could aid in treating or preventing HIV-associated peripheral neuropathy.

Investigating the Presence of Rotavirus in Wastewater Samples of Bhopal Region, India, by Utilizing Droplet Digital Polymerase Chain Reaction

Investigating the Presence of Rotavirus in Wastewater Samples of Bhopal Region, India, by Utilizing Droplet Digital Polymerase Chain Reaction

Optimal design and implement of a droplet generator of droplet digital polymerase chain reaction via taguchi method

Optimal design and implement of a droplet generator of droplet digital polymerase chain reaction via taguchi method

Origin of pathogenic variant and mosaicism in families with a sporadic case of haemophilia B.

Of newly diagnosed cases of haemophilia B, the proportion of sporadic cases is usually 50% of severe cases and 25% of moderate/mild cases. However, cases presumed to be sporadic due to family history may not always be sporadic. Few case reports have been published on mosaicism in haemophilia B. The present study aimed to trace the origin of the pathogenic variant in a well-defined cohort of sporadic cases of haemophilia B by haplotyping markers. It also aimed to determine the frequency of mosaicism in presumed non-carrier mothers. The study group was 40 families, each with a sporadic case of haemophilia B analysed in two-to-three generations by Sanger sequencing, haplotyping and using the sensitive droplet digital polymerase chain reaction (ddPCR) technique. In 31/40 (78%) of the families, the mother carried the same pathogenic variant as her son, while Sanger sequencing showed that 9/40 (22%) of the mothers did not carry this variant. Of these variants, 2/9 (22%) were shown to be mosaics by using the ddPCR technique. 16/21 carrier mothers, with samples from three generations available, had a de novo pathogenic variant of which 14 derived from the healthy maternal grandfather. The origin of the pathogenic variant in sporadic cases of haemophilia B is most often found in the X-chromosome derived from the maternal grandfather or, less often, from the maternal grandmother. Mosaic females seem to be found at the same frequency as in haemophilia A but at a lower percentage of the pathogenic variant.

Acalabrutinib, venetoclax, and obinutuzumab in relapsed/refractory CLL: final efficacy and ctDNA analysis of the CLL2-BAAG trial

AbstractThe phase 2 CLL2-BAAG trial tested the measurable residual disease (MRD)–guided triple combination of acalabrutinib, venetoclax, and obinutuzumab after optional bendamustine debulking in 45 patients with relapsed/refractory chronic lymphocytic leukemia (CLL; 1 patient was excluded from the analysis because of a violation of exclusion criteria). MRD was measured by flow cytometry (FCM; undetectable MRD <10–4) in peripheral blood (PB) and circulating tumor DNA (ctDNA) using digital droplet polymerase chain reaction of variable-diversity-joining (VDJ) rearrangements and CLL-related mutations in plasma. MRD recurrence was defined as detectable ctDNA and/or MRD ≥10–4 after achieving both undetectable MRD (uMRD) and undetectable ctDNA. The median number of previous treatments was 1 (range, 1-4); 18 patients (40%) had received a Bruton tyrosine kinase inhibitor (BTKi) and/or venetoclax before inclusion, 14 of 44 (31.8%) had TP53 aberrations, and 34 (75.6%) had unmutated IGHV. With a median observation time of 36.3 months and all patients off-treatment for a median of 21.9 months, uMRD <10–4 in PB was achieved in 42 of the 45 patients (93.3%) at any time point, including 17 of 18 (94.4%) previously exposed to venetoclax/BTKi and 13 of 14 (92.9%) with TP53 aberrations. The estimated 3-year progression-free and overall survival rates were 85.0% and 93.8%, respectively. Overall, 585 paired FCM/ctDNA samples were analyzed and 18 MRD recurrences (5 with and 13 without clinical progression) occurred after the end of treatment. Twelve samples were first detected by ctDNA, 3 by FCM, and 3 synchronously. Patients with earlier detection of ctDNA appeared to have a genetically higher risk of disease. In conclusion, time-limited MRD-guided acalabrutinib, venetoclax, and obinutuzumab achieved deep remissions in almost all patients with relapsed/refractory CLL. The addition of ctDNA-based analyses to FCM MRD assessment seems to improve early detection of relapses. This trial was registered at www.ClinicalTrials.gov #NCT03787264.

Low C4A copy numbers and higher HERV gene insertion contributes to increased risk of SLE, with absence of association with disease phenotype and disease activity.

Low copy numbers (CNs) of C4 genes are associated with systemic autoimmune disorders and affects autoantibody diversity and disease subgroups. The primary objective of this study was to characterize diversity of complement (C4) and C4-Human Endogenous Retrovirus (HERV) gene copy numbers in SLE. We also sought to assess the association of C4 and C4-HERV CNs with serum complement levels, autoantibodies, disease phenotypes and activity. Finally, we checked the association of C4 and HERV CNs with specific HLA alleles. Genomic DNA from 70 SLE and 90 healthy controls of south Indian Tamil origin were included. Demographic, clinical and serological data was collected in a predetermined proforma. CNs of C4A and C4B genes and the frequency of insertion of 6.4kb HERV within C4 gene (C4AL, C4BL) was determined using droplet digital polymerase chain reaction (ddPCR). A four digit high resolution HLA genotyping was done using next generation sequencing. In our cohort, the total C4 gene copies ranged from 2 to 6. Compared to controls, presence of two or less copies of C4A gene was associated with SLE risk (p = 0.005; OR = 2.79; 95% CI = 1.29-6.22). Higher frequency of HERV insertion in C4A than in C4B increases such risk (p = 0.000; OR = 12.67; 95% CI = 2.80-115.3). AL-AL-AL-BS genotype was significantly higher in controls than SLE (9%vs1%, p = 0.04; OR = 0.15, 95% CI = 0.00-0.16). Distribution of HLA alleles was not different in SLE compared to controls as well as in SLE subjects with ≤ 2 copies and > 2 copies of C4A, but HLA allele distribution was diverse in subjects with C4B ≤ 2 copies and > 2 copies. Finally, there was no correlation between the C4 and the C4-HERV diversity and complement levels, autoantibodies, disease phenotypes and activity. In conclusion, our data show that, low C4A copy number and higher insertion of HERV-K in C4A increases the risk for SLE. C4 and C4-HERV CNs did not correlate with serum complements, autoantibodies, disease phenotypes and activity in SLE. Further validation in a larger homogenous SLE cohort is needed.

Genomic and Pathologic Profiling of Very Well-Differentiated Gastric Adenocarcinoma of Intestinal Type: A Study With Emphasis on Diffuse-Type Transformation.

Very well-differentiated adenocarcinoma of intestinal type is a distinct subtype of gastric cancer characterized by anastomosing glands with a hand-in-hand pattern and low-grade cytologic atypia resembling intestinal metaplasia. This is a slow-growing neoplasm with an indolent clinical course; however, a subset demonstrates transformation into adenocarcinoma with higher-grade histology, typically diffuse-type carcinoma, and behaves aggressively. This study aimed to better characterize the genomic and pathologic features, with a focus on factors associated with diffuse-type transformation. A total of 58 cases with (n=31) and without (n=27) diffuse-type transformation were analyzed for molecular and pathologic features. First, comprehensive deep DNA sequencing was conducted in 18 cases (discovery cohort), followed by a digital droplet polymerase chain reaction of hot spot RHOA mutations in 40 cases (validation cohort). In total, RHOA mutations were the most common alteration (34%), followed by loss of ARID1A (12%), p53 alterations (10%), and CLDN18::ARHGAP26/6 fusions (3.4%). FGFR2 amplification was identified in an advanced case with a p53 alteration. Altered p53 expression was recognized only in higher-grade components and was significantly associated with advanced disease (P=0.0015) and diffuse-type transformation (P=0.026). A mixed mucin phenotype was also strongly correlated with advanced disease (P<0.001) and diffuse-type transformation (P<0.001). Decreased E-cadherin expression was frequently observed (74%) in poorly cohesive components. This study demonstrated that a subset of RHOA-mutant diffuse-type gastric cancers develops through the transformation of very well-differentiated adenocarcinoma of intestinal type. Our observations suggest a mixed mucin phenotype as a risk factor and alterations in p53 and E-cadherin as drivers of diffuse-type transformation.

Identification of a mosaic MTOR variant in purified neuronal DNA in a patient with focal cortical dysplasia using a novel depth electrode harvesting technique.

Recent studies have identified brain somatic variants as a cause of focal epilepsy. These studies relied on resected tissue from epilepsy surgery, which is not available in most patients. The use of trace tissue adherent to depth electrodes used for stereo electroencephalography (EEG) has been proposed as an alternative but is hampered by the low cell quality and contamination by nonbrain cells. Here, we use our improved depth electrode harvesting technique that purifies neuronal nuclei to achieve molecular diagnosis in a patient with focal cortical dysplasia (FCD). Depth electrode tips were collected, pooled by brain region and seizure onset zone, and nuclei were isolated and sorted using fluorescence-activated nuclei sorting (FANS). Somatic DNA was amplified from neuronal and astrocyte nuclei using primary template amplification followed by exome sequencing of neuronal DNA from the affected pool, unaffected pool, and saliva. The identified variant was validated using droplet digital polymerase chain reaction (PCR). An 11-year-old male with drug-resistant genetic-structural epilepsy due to left anterior insula FCD had seizures from age 3 years. Stereo EEG confirmed seizure onset in the left anterior insula. The two anterior insula electrodes were combined as the affected pool and three frontal electrodes as the unaffected pool. FANS isolated 140 neuronal nuclei from the affected and 245 neuronal nuclei from the unaffected pool. A novel somatic missense MTOR variant (p.Leu489Met, CADD score 23.7) was identified in the affected neuronal sample. Droplet digital PCR confirmed a mosaic gradient (variant allele frequency = .78% in affected neuronal sample; variant was absent in all other samples). Our findings confirm that harvesting neuronal DNA from depth electrodes followed by molecular analysis to identify brain somatic variants is feasible. Our novel method represents a significant improvement compared to the previous method by focusing the analysis on high-quality cells of the cell type of interest.

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Efficacy of third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in advanced NSCLC with different T790M statuses tested via digital droplet polymerase chain reaction ddPCR and next-generation sequencing

ABSTRACT Objectives We hypothesize that digital droplet polymerase chain reaction (ddPCR) would optimize the treatment strategies in epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitors (TKIs) relapsed patients. In this study, we compared the efficacy of third-generation TKIs with various T790M statuses via ddPCR and next-generation sequencing (NGS). Methods NGS was performed on blood samples of patients progressed from previous EGFR-TKIs for resistance mechanism. T790M-negative patients received further liquid biopsy using ddPCR for T790M detection. Results A cohort of 40 patients were enrolled, with 30.0% (12/40) T790M-positive via NGS (Group A). In another 28 T790M-negative patients by NGS, 11 (39.3%) were T790M-positive (Group B) and 17 (60.7%) were T790M-negative (Group C) via ddPCR. A relatively longer progression-free survival (PFS) was observed in group A (NR) and group B (10.0 months, 95% CI 7.040-12.889) than in group C (7.0 months, 95%CI 0.000-15.219), with no significant difference across all three groups (p = 0.196), or between group B and C (p = 0.412). EGFR-sensitive mutation correlated with inferior PFS (p = 0.041) and ORR (p = 0.326), and a significantly lower DCR (p = 0.033) in T790M-negative patients via NGS (n = 28). Conclusion This study indicates that ddPCR may contribute as a supplement to NGS in liquid biopsies for T790M detection in EGFR-TKIs relapsed patients and help to optimize the treatment strategies, especially for those without coexistence of EGFR-sensitive mutation. Trial registration www.clinicaltrials.gov identifier is NCT05458726.

Pursuing dynamics of minimal residual leukemic subclones in relapsed and refractory acute myeloid leukemia during conventional therapy.

Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied. Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored. Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse. In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

Prognostic value of poly-microorganisms detected by droplet digital PCR and pathogen load kinetics in sepsis patients: a multi-center prospective cohort study.

This study aimed to investigate the prognostic value of a novel droplet digital polymerase chain reaction (DDPCR) assay in sepsis patients. In this prospective cohort study, univariable and multivariable Cox regressions were used to assess risk factors for 28-day mortality. We also monitored pathogen load together with clinical indicators in a subgroup of the cohort. A total of 107 sepsis patients with positive baseline DDPCR results were included. Detection of poly-microorganisms [adjusted hazard ratio (HR) = 3.19; 95% confidence interval (CI) = 1.34-7.62; P = 0.009], high Charlson Comorbidity Index (CCI) score (adjusted HR = 1.14; 95% CI = 1.01-1.29; P = 0.041), and Sequential Organ Failure Assessment (SOFA) score (adjusted HR = 1.18; 95% CI = 1.05-1.32; P = 0.005) at baseline were independent risk factors for 28-day mortality while initial pathogen load was not associated (adjusted HR = 1.17; 95% CI = 0.82-1.66; P = 0.385). Among 63 patients with serial DDPCR results, an increase in pathogen load at days 6-8 compared to baseline was a risk factor for 28-day mortality (P = 0.008). Also, pathogen load kinetics were significantly different between day-28 survivors and nonsurvivors (P = 0.022), with a decline overtime only in survivors and an increase from days 3 and 4 to days 6-8 in nonsurvivors. Using DDPCR technique, we found that poly-microorganisms detected and increased pathogen load a week after sepsis diagnosis were associated with poor prognosis.IMPORTANCEThis prospective study was initiated to explore the prognostic implications of a novel multiplex PCR assay in sepsis. Notably, our study was the largest cohort of sepsis with droplet digital polymerase chain reaction pathogen monitoring to date, allowing for a comprehensive evaluation of the prognostic significance of both pathogen species and load. We found that detection of poly-microorganisms was an independent risk factors for 28-day mortality. Also, pathogen load increase 1 week after sepsis diagnosis was a risk factor for 28-day mortality, and differential pathogen load kinetics were identified between day-28 survivors and nonsurvivors. Overall, this study demonstrated that pathogen species and load were highly correlated with sepsis prognosis. Patients exhibiting conditions mentioned above face a more adverse prognosis, suggesting the potential need for an escalation of antimicrobial therapy.Registered at ClinicalTrials.gov (NCT05190861).

Abstract 2498: Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue

Abstract Introduction Tumor profiling including RAS, BRAF, HER2, and MSI/MMR status, is required to determine the treatment for patients with metastatic colorectal cancer (mCRC) at the time of diagnosis. While comprehensive tumor profiling using tissue-based next-generation sequencing (NGS) is increasingly adopted for initial profiling in mCRC, the role of circulating tumor DNA (ctDNA) NGS as initial testing in patients with sufficient tumor tissue is not clearly understood. We assessed the clinical value of ctDNA sequencing compared to tumor NGS in patients with newly diagnosed mCRC who have sufficient tumor specimens. Methods We prospectively enrolled consecutive patients with newly diagnosed mCRC at the National Cancer Center Korea. As per the institutional protocol for mCRC, initial tumor profiling was performed on primary tumor tissue using an in-house NGS panel (NCC PCP ver.3), which included 525 genes. For ctDNA sequencing, patients were evaluated using the AlphaLiquid®100 comprehensive cancer panel (IMBdx, Inc.), which included 118 genes, before the initiation of chemotherapy. Additionally, immunohistochemical (IHC) testing for HER2 and polymerase chain reaction (PCR)/IHC testing for MSI and/or MMR were performed to assess the accuracy of HER2 and MSI/MMR status. Results A total of 188 patients were enrolled. In 139 eligible patients, 275 potentially actionable mutations were found in 12 selected CRC-related genes (APC, TP53, KRAS, NRAS, BRAF, FBXW7, PIK3CA, ERBB2, SMAD4, NF1, EGFR, MET). Of these, 32% were found both in ctDNA and tissue; 54% were found in ctDNA only; 12% in tissue only, and 2% were discordant. For RAS/BRAF mutations, which are required for anti-EGFR treatment decisions, the concordance rate between ctDNA and tissue NGS was 83.1%, and the concordance was higher in patients with higher ctDNA concentrations. For 9 patients with potentially actionable copy number variations (CNV) in EGFR, HER2, MET, and FGFR1, 3 cases were found by both assays; 4 were found by ctDNA only, and 2 were found by tissue only. ctDNA NGS correctly predicted MSI/MMR status in 2 out of 4 patients with MSI-H/dMMR; in the 2 other patients, the MSI and MMR statuses were different, suggesting potential false positivity. In addition, the fold changes in ctDNA dynamics during treatment significantly correlated with changes in tumor size and CEA levels, as well as with droplet digital PCR copy number fold changes. In a patient with MET amplification, ctDNA NGS identified MET Y1230H as a potential acquired resistance mutation after crizotinib treatment, which responded to cabozantinib but not to capmatinib. Conclusions Initial tumor profiling using ctDNA NGS yielded outcomes comparable to those of tumor tissue NGS in guiding treatment for patients with newly diagnosed mCRC, thereby suggesting its utility as an initial profiling method in mCRC. Citation Format: Yongjun Cha, Bun Kim, Dong Woon Lee, Hyun Yang Yeo, Chang Won Hong, Kyung Su Han, Byung Chang Kim, Hee Jin Chang. Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2498.

Leveraging wastewater surveillance to actively monitor Covid‐19 community dynamics in rural areas with reduced reliance on clinical testing

AbstractThe prevalence of coronavirus disease 2019 (Covid‐19) in the community has become more difficult to gauge utilizing clinical testing due to a decrease in reported test results stemming from the availability of at‐home test kits and a reduction in the number of cases seeking medical treatment. The purpose of this study was to examine the trend of diminishing correlation between reported clinical cases of Covid‐19 and wastewater‐based surveillance epidemiological data as home testing became available in the Eastern Upper Peninsula of Michigan. Wastewater grab samples were collected weekly from 16 regional locations from June 2021 to December 2022. Samples were analyzed for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) N1 and N2 viral particles using reverse transcriptase digital droplet polymerase chain reaction (RT ddPCR). N1 and N2 gene copies were correlated with clinical cases. The t test was used to determine the correlation deterioration point. Clinical cases postdeterioration were calculated for high‐correlated predeterioration locations using linear regression. Correlation between the wastewater‐based surveillance of SARS‐CoV‐2 and reported clinical cases deteriorated after February 1, 2022. This corresponds with the timeframe in which commercially available at‐home test kits became available in the United States. The increase in at‐home testing for SARS‐CoV‐2 likely contributed to the decrease in reported clinical positive tests in early 2022, providing an unrealistic picture of the presence of Covid‐19 in the community. As measures to reduce exposure such as personal masking, clinical testing, social isolating, and quarantining continue to decline, wastewater surveillance for the presence of SARS‐CoV‐2 may be the best method for public health professionals to remain aware of virus dynamics in localized regions. Time‐series modeling adds another layer of information when clinical data is unobtainable or underreported.

Optimal filter materials for protist quantification via droplet digital PCR

The use of droplet digital polymerase chain reaction (ddPCR) has greatly improved the quantification of harmful protists, outperforming traditional methods like quantitative PCR. Notably, ddPCR provides enhanced consistency and reproducibility at it resists PCR inhibitors commonly found in environmental DNA samples. This study aimed to determine the most effective filter material for ddPCR protocols by assessing the reproducibility of species-specific gene copy numbers and filtration time across six filter types: cellulose acetate (CA), mixed cellulose ester (MCE), nylon (NY), polycarbonate (PC), polyethersulfone (PES), and polyvinylidene fluoride (PVDF). The study used two species of Chattonella marina complexes as a case study. Filtration rates were slower for NY, PC, and PVDF filters. Moreover, MCE, NY, PES, and PVDF yielded lower DNA amounts than other filters. Importantly, the CA filter exhibited the lowest variance (38–39%) and the highest determination coefficients (R2 = 0.92–0.96), indicating superior performance. These findings suggest that the CA filter is the most suitable for ddPCR quantification of marine protists, offering quick filtration and reliable reproducibility.