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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., Peripheral blood HIV DNA levels from 164 South African in utero HIV-infected infants showed that with antiretroviral mother-to-child transmission prophylaxis, combination antiretroviral therapy initiation timing in the first 3 weeks of life does not significantly influence latent HIV reservoir size.

Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

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.

Detection and quantification of tumor-derived mutations in cell-free DNA (cfDNA) holds great potential for non-invasive tumor diagnostics and molecular monitoring. We developed highly sensitive discriminatory multi-target digital droplet PCR (ddPCR) assays covering 14 KRAS and NRAS hotspot mutations.

Detection and quantification of tumor-derived KRAS and NRAS mutations in plasma cell-free DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high sensitivity, specificity, robustness, and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized, and clinically validated mutation-specific locked nucleic acid–based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into seven discriminatory multitarget screening assays covering two to six single-nucleotide variants each. Limit of detection, limit of blank, and interassay accuracy were determined. Assay performance and suitability for screening in cfDNA were validated with plasma samples from a clinically fully characterized cohort of pancreatic cancer patients and healthy controls. Limits of detection for single-target assays were between 0.0015% and 0.069% variant allele fraction, and between 0.022% and 0.16% for multitarget assays. Dilution linearity and interassay accuracy were excellent throughout (r2 > 0.99). Multitarget assay screening of cfDNA extracted from pancreatic cancer patients with unknown KRAS mutational status correctly identified single-nucleotide variants in 45 of 45 (100%) of tumor-derived cell-free DNA–positive samples. In summary, we herein present and clinically validate generic single-target and discriminatory multitarget ddPCR assays for KRAS and NRAS hot spot mutations with broad applicability for clinical and translational research.

Background UVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology. Study design and methods Samples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out-of-specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross-validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed. Results Apheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single-nucleotide polymorphisms in the primer- and probe-binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra- and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment. Conclusion The ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC-treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.

BACKGROUND: Inversions involving intron 22 (Inv22) of F8 are detected in approximately 45% of all severe hemophilia A patients. Diagnosis is complicated by the large size of the ~9.5 kb int22h repeated sequence which generates the inversions. Methods such as long-range PCR and inverse-shifting PCR are currently used diagnostically, but suffer from low PCR efficiencies and are difficult to standardize.OBJECTIVES: To design and validate a sensitive and robust assay for the detection of F8 int22h inversions.METHODS: Digital droplet PCR using mile-post assays was used to investigate archival DNA samples.RESULTS: The detection of linkage as a function of physical distance between loci was investigated using an anchor locus and mile-post loci located at 1, 6, 12 and 15 kb distances from the anchor locus. The proportion of linked molecules decreased with increasing distance between loci and showed 30-40% linked molecules for loci 12-15 kb apart. Mile-post assays specific for wild type and Inv22 type 1 and 2 chromosomes were then designed and optimized. All three assays showed high specificities and sensitivities, with coefficients of variation < 5% for all assays. Analysis of 106 patients and 20 carrier mothers showed complete concordance with previously known mutation status. The analysis demonstrated the robustness of the assays versus input DNA concentration (6 ng and higher) and level of fragmentation.CONCLUSIONS: Digital droplet PCR and mile-post assays can be used to detect F8 int22h inversions. The assay systems are technically simple to perform, highly efficient and robust. (Less)

The "sample-to-answer" integration and automation of circulating tumor DNA (ctDNA)-based liquid biopsy using digital PCR (dPCR) has been hampered by the complicated operations of liquids with volumes ranging from milliliter samples to nanoliter droplets. On the basis of a "3D extensible" design paradigm proposed previously, an integrated droplet digital PCR (IddPCR) microdevice was successfully developed to automate the entire process of liquid biopsy, from the extraction of ctDNA in 2 mL of plasma using magnetic beads to the generation, amplification, and screening of over 30 000 droplets for detection. A series of reagent mixing structures, including macro-, meso-, and micromixers, was designed to enable efficient reagent handling and mixing at different volume scales. The volume thresholds of the microscale and macroscale in the IddPCR device were calculated to be 40 and 100 μL, respectively, based on the fluid dynamics and sizes of the device structures, so that different mixers can be selected according to the reagent volumes. The DNA extraction efficiency obtained on the device was determined to be ∼60%, and the on-chip ddPCR demonstrated a high correlation with an

Marzipan is a mixture of almonds, sugar, and water. Almonds can be replaced by apricot kernels, which results in a similar product called persipan. Depending on the prices of almonds, profit can be maximized using apricot kernels instead of almonds. If not specified on the product, this kind of substitution is illegal and is prosecuted by official food control authorities. Likewise, also commercial buyers would like to know which product they bought. Real-time PCR systems for the determination of apricot DNA are already available, however, real-time PCR requires the use of external standards, which have a direct impact on the accuracy of the measurement. Currently, such standards are not available. In contrast to real-time PCR, digital PCR does not require external standards and exhibits a smaller measurement uncertainty as shown in recent publications. Therefore, we developed a duplex droplet digital PCR system to measure the proportion of apricot in marzipan without the use of external standards. We present validation data and results of an international proficiency test, underlining the applicability of this system.

Background: Circulating tumor DNA (ctDNA) is found in the plasma of over 90% of patients with advanced breast cancer (BC). ctDNA analysis can establish the current genomic profile of an individual’s cancer and identify potentially targetable mutations. The genomic landscape of advanced BC and clinical associations has yet to be fully defined. This analysis describes the genomic landscape of ctDNA in patients screened for the UK plasmaMATCH study. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. The trial consists of parallel treatment cohorts (Cohorts A-E) with therapies matched to mutations identified in ctDNA testing. The genomic landscape of ctDNA in advanced BC was prospectively analysed for hotspot PIK3CA,AKT1, HER2 and ESR1 mutations using digital droplet PCR (ddPCR, Bio-Rad), and using an error-corrected 73-gene targeted panel (Guardant360, Guardant Health) prospectively from part-way through the trial, and retrospectively for the remaining patients. Results: Entry into ctDNA testing for Cohorts A-D was closed on 26/Apr/2019, at which time 1044 patients had been registered and 1025 tested with ddPCR. 800 patients had targeted panel ctDNA sequencing (364 prospective, 436 retrospective) (Table 1). Targeted sequencing identified a somatic alteration in 92.9% of patients (743/800). The most frequent mutations were TP53 (44.1%), PIK3CA (34.9%), ESR1 (33.1%), GATA3 (11.0%) and ARID1A (7.8%). ESR1 (33.1%) and KRAS (4.1%) alterations occurred at higher frequency in ctDNA compared to a metastatic tissue sequencing series (q Genomic AlterationddPCR*Targeted Panel**All % n = 1025HR+ HER2-% n = 658HR+ HER2+ % n = 62HR- HER2+ % n = 36TNBC % n = 172All % n = 800HR+ HER2- % n = 515HR+ HER2+ % n = 48HR- HER2+ % n = 26TNBC % n = 136PIK3CA mutation25.829.933.919.49.334.939.041.734.614.7ESR1 mutation27.737.819.40033.143.522.900.7AKT1 mutation4.24.01.604.75.05.22.104.4ERBB2 mutation2.73.208.31.26.46.612.515.42.2PTEN mutationNANANANANA6.98.54.23.84.4ERBB2 amplificationNANANANANA5.81.931.357.71.5MSI highNANANANANA1.10.42.101.5* Phenotype data based on 928 patients with known phenotype tested by ddPCR for targetable hotspot mutations.** Phenotype data based on 725 patients with known phenotype sequenced by targeted panel. All pathogenic mutations are shown, including mutations not tested by ddPCR. Citation Format: Belinda Kingston, Hannah Bye, Michael Hubank, Giselle Walsh, Claire Swift, Matthew Beaney, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Richard D Baird, Rebecca Roylance, Katie Wilkinson, Isaac Garcia-Murillas, Judith M Bliss, Nicholas Turner, Alistair Ring, on behalf of the plasmaMATCH Trial Management Group. The genomic landscape of breast cancer based on ctDNA analysis: Data from the plasmaMATCH trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS3-07.

Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.

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, kappa = 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.

Abnormal genetic and epigenetic modifications play a key role in esophageal cancer. By Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq), this study compared chromatin accessibility landscapes among two esophageal squamous cell carcinoma (ESCC) cell lines, KYSE-30 and KYSE-150, and a non-cancerous esophageal epithelial cell line, HET-1A. Data showed that hyper-accessible regions in ESCC cells contained genes related with cancer hallmarks, such as epidermal growth factor receptor (EGFR). Multi-omics analysis and digital-droplet PCR results demonstrated that several non-coding RNAs in EGFR upstream were upregulated in ESCC cells. Among them, one appeared to act as an enhancer RNA responsible for EGFR overexpression. Further motif analysis and pharmacological data suggested that AP-1 family transcription factors were able to bind the hyper-accessible regions and thus to regulate cancer cell proliferation and migration. This study discovered a putative enhancer RNA for EGFR gene and the reliance of ESCC on AP-1 transcription factor.

Here, we compare the number of mutant molecules and the variant allele frequency as units of measurement for circulating tumor DNA. We conclude that when using digital droplet PCR, both units of measurement yield greater agreement than when using next generation sequencing. Low frequent mutations and molecular coverage are important factors affecting agreement. These results will improve circulating tumor DNA analysis, contributing to better cancer patient management., 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.

While tissue biopsy has for the longest time been the gold-standard in biomedicine, precision/personalized medicine is making the shift toward liquid biopsies. Cell-free DNA (cfDNA) based genetic and epigenetic biomarkers reflect the molecular status of its tissue-of-origin allowing for early and non-invasive diagnostics of different pathologies. However, selection of preanalytical procedures (including cfDNA isolation) as well as analytical methods are known to impact the downstream results. Calls for greater standardization are made continuously, yet comprehensive assessments of the impact on diagnostic parameters are lacking. This study aims to evaluate the preanalytic and analytic factors that influence cfDNA diagnostic parameters in blood and semen. Text mining analysis has been performed to assess cfDNA research trends, and identify studies on isolation methods, preanalytical and analytical impact. Seminal and blood plasma were tested as liquid biopsy sources. Traditional methods of cfDNA isolation, commercial kits (CKs), and an in-house developed protocol were tested, as well as the impact of dithiothreitol (DTT) on cfDNA isolation performance. Fluorimetry, qPCR, digital droplet PCR (ddPCR), and bioanalyzer were compared as cfDNA quantification methods. Fragment analysis was performed by qPCR and bioanalyzer while the downstream application (cfDNA methylation) was analyzed by pyrosequencing. In contrast to blood, semen as a liquid biopsy source has only recently begun to be reported as a liquid biopsy source, with almost half of all publications on it being review articles. Experimental data revealed that cfDNA isolation protocols give a wide range of cfDNA yields, both from blood and seminal plasma. The addition of DTT to CKs has improved yields in seminal plasma and had a neutral/negative impact in blood plasma. Capillary electrophoresis and fluorometry reported much higher yields than PCR methods. While cfDNA yield and integrity were highly impacted, cfDNA methylation was not affected by isolation methodology or DTT. In conclusion, NucleoSnap was recognized as the kit with the best overall performance. DTT improved CK yields in seminal plasma. The in-house developed protocol has shown near-kit isolation performance. ddPCR LINE-1 assay for absolute detection of minute amounts of cfDNA was established and allowed for quantification of samples inhibited in qPCR. cfDNA methylation was recognized as a stable biomarker unimpacted by cfDNA isolation method. Finally, semen was found to be an abundant source of cfDNA offering potential research opportunities and benefits for cfDNA based biomarkers development related to male reproductive health., Graphical Abstract Depiction of the experimental design.

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.

We describe the establishment of a seronegative occult hepatitis B virus (HBV) infection (OBI) in a successfully vaccinated infant who underwent liver transplantation from an donor positive for antibody to hepatitis B core antigen (anti-HBc). The use of highly sensitive droplet digital polymerase chain reaction assays revealed a not negligible and transcriptionally active intrahepatic HBV reservoir (circular covalently closed DNA, relaxed circular DNA, and pregenomic RNA: 5.6, 2.4, and 1.1 copies/1000 cells, respectively), capable to sustain ongoing viral production and initial liver damage. Next-generation sequencing revealed a peculiar enrichment of hepatitis B surface antigen vaccine-escape mutations that could have played a crucial role in OBI transmission. This clinical case highlights the pathobiological complexity and the diagnostic challenges underlying OBI.

Since 2000, we have investigated 67 consecutive patients with stage I/II follicular lymphoma (FL) for the presence of BCL2/IGH rearrangements by polymerase chain reaction (PCR), real time quantitative PCR (RQ-PCR) and digital droplet PCR (ddPCR). All patients were treated with involved-field radiotherapy (IF-RT) (24-30 Gy). From 2005, patients with minimal residual disease (MRD) after IF-RT received rituximab (R) (375 mg/m2 , 4 weekly administrations). The median follow-up is 82 months (17-196). At diagnosis, 72% of patients were BCL2/IGH+. Progression-free survival (PFS) was significantly better in patients with undetectable/low levels (

Background: Non-classical HER2 FISH results were recently reclassified in the 2018 HER2 guidelines update, and concurrent IHC testing was recommended as part of additional workup to determine the final HER2 status in these groups. In this study, we explored the genomic landscape of HER2 FISH groups using digital droplet PCR (ddPCR) and targeted next-generation sequencing (NGS) on invasive breast carcinomas. Methods: Fifty-one clinical samples with HER2 FISH and IHC results were included in our analysis and classified into FISH groups based on the updated 2018 ASCO/CAP HER2 testing guideline: (i) Group 1A with ratio ≥2 and signals/cell ≥6, (ii) Group 1B with ratio ≥2 and signals/cell ≥4 and 50% of exons). Results: Mean ddPCR ratios varied amongst the different FISH groups (P < 0.0001). As expected, patients with Group 1A had the highest mean ddPCR ratios compared to those with other FISH findings (P < 0.0001). Furthermore, there was a correlation between ERBB2 ddPCR ratios and HER2 FISH ratios (ρe19 = 0.4435, P = 0.001 and ρe21 = 0.4644, P = 0.0006). Using ddPCR, ERBB2 amplifications were detected in all classically amplified Group 1A cases (5/5) and in none of the classically non-amplified Group 5 cases (0/12). Interestingly, ddPCR assays called ERBB2 amplification in four cases with non-classical results: one in Group 2 (1/6), two in Group 3 (2/6), and one in Group 4 (1/17), including two cases in Groups 3 and 4 which also showed concomitant HER2 overexpression by IHC (3+). Similarly, targeted NGS revealed ERBB2 amplification in all Group 1A cases (5/5) and in none of the Group 5 cases (0/12). Furthermore, NGS detected amplification in three non-classical cases: one in Group 1B (1/5), one in Group 3 (1/6), and one in Group 4 (1/17), including one case in Group 1B which was not called amplified by ddPCR. Notably, the three cases with amplification by NGS were the only three cases in the non-classical groups with HER2 overexpression by IHC. Overall, there was a strong concordance between ERBB2 amplification status by ddPCR/NGS and HER2 overexpression by IHC (κe19 = 0.79, κe21 = 0.92, κNGS = 1.0). Conclusion: ERBB2 amplification using ddPCR and NGS is correlated with HER2 overexpression in both classical and non-classical FISH groups, thus providing genomic evidence to support the recent recommendation for concurrent IHC testing in cases with unusual FISH results. Our findings also highlight a potential role of ddPCR and targeted NGS in the workup of challenging HER2 cases. Citation Format: Yang S-R, Bouhlal Y, De La Vega FM, Ballard M, West RB, Sibley RK, Kuo CJ, Vilborg A, Allison KH. ERBB2 copy number analysis of invasive breast carcinoma using digital droplet PCR and targeted next-generation sequencing: A focus on 'non-classical' HER2 FISH groups using the 2018 ASCO/CAP HER2 testing guideline [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-12.

A value was assigned in 2009 to the Legionella DNA Certified Reference Material, and the stability study conducted using quantitative PCR found a low level of degradation. Herein, the Digital Droplet PCR method for Legionella DNA was qualified and used to provide absolute quantification of the CRM.

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.

Background: Ureaplasma spp. are association with a various of infectious diseases in female, but it still limited evidence for the pathogenicity in nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for quantified the load of Ureaplasma spp in cervical swabs. Methods: A total of 293 non-specific cervicitis (NSC) patients and 211 asymptomatic female fulfilled the inclusion criteria. Cervical swabs were identified by qPCR and further absolutely quantified by ddPCR. Results: The prevalence of U.parvum were 51.9% (152/293) and 46.9% (99/211); while U.urealyticum were 8.2% (24/293) and 8.1% (17/211) in the NSC and Control group, respectively. In addition, the average Ct value and median copy number per microliter of U.parvum were 31.33 (n=152) and 599 (n=48) in the NSC group and 33.68 (n=99) and 17.4 (n=33) in control group, respectively, suggest that the load of U.parvum of NSC group were significantly higher than the asymptomatic individual (P0.05). Conclusions: our study suggests that often carrying U.parvum at a high load but not U.urealyticum may have an important implications on the development and progression of cervicitis among female.

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

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.

Controlling and monitoring the still ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic regarding geographical distribution, evolution, and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and sharing sequence data worldwide. Efficient sequencing strategies enable the retrieval of increasing numbers of high-quality, full-length genomes and are, hence, indispensable. Two opposed enrichment methods, tiling multiplex PCR and sequence hybridization by bait capture, have been established for SARS-CoV-2 sequencing and are both frequently used, depending on the quality of the patient sample and the question at hand. Here, we focused on the evaluation of the sequence hybridization method by studying five commercially available sequence capture bait panels with regard to sensitivity and capture efficiency. We discovered the SARS-CoV-2-specific panel of Twist Bioscience to be the most efficient panel, followed by two respiratory panels from Twist Bioscience and Illumina, respectively. Our results provide on the one hand a decision basis for the sequencing community including a computation for using the full capacity of the flow cell and on the other hand potential improvements for the manufacturers. IMPORTANCE Sequencing the genomes of the circulating SARS-CoV-2 strains is the only way to monitor the viral spread and evolution of the virus. Two different approaches, namely, tiling multiplex PCR and sequence hybridization by bait capture, are commonly used to fulfill this task. This study describes for the first time a combined approach of droplet digital PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were able to determine the most sensitive and efficient capture panel, distinguish the mode of action of the various bait panels, and compute the number of read pairs needed to recover a high-quality full-length genome. By calculating the minimum number of read pairs needed, we are providing optimized flow cell loading conditions for all sequencing laboratories worldwide that are striving for maximizing sequencing output and simultaneously minimizing time, costs, and sequencing resources.

We present a patient who had two allogeneic bone marrow transplantations for acute lymphocytic leukemia. She developed slowly progressive limb-girdle weakness in the context of other symptoms of graft-vs.-host disease (GVHD). Her myopathy symptoms had been initially attributed to GVHD, but when she progressed despite immunotherapy, genetic testing was requested. Initial testing was performed on a blood sample, identifying a variant of unknown significance in DMD. Subsequent testing of DNA from the patient's muscle tissue identified two pathogenic variants in CAPN3, with absence of the DMD variant (this latter variant presumed to have been received from the donor). Allele-specific digital droplet qPCR permitted the quantification of the donor variant in various tissues from the patient (whole skin, isolated fibroblasts, whole blood, saliva, buccal cells, urine sediment, and two muscle biopsies taken at a 2 year interval). This report emphasizes that genetic disease should still be considered in the context of presumably acquired disease, and also demonstrates the extent of transdifferentiation of donor cells into other tissues.

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 log(10) 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.

BackgroundCurrent next generation sequencing (NGS) and microarray based Non-Invasive Prenatal Tests (NIPT), used for the detection of common fetal trisomies, are still expensive, time consuming and need to be performed in centralized laboratories. To improve NIPT in clinical routine practice as universal prenatal screening, we have developed a digital droplet PCR (ddPCR) based assay called iSAFE NIPT using cell free fetal DNA (cffDNA) for detection of fetal trisomies 13, 18 and 21 in a single reaction with advantage of high diagnostic accuracy and reduced cost.Materials and MethodsWe first used artificial DNA samples to evaluate analytical sensitivity and specificity of the iSAFE NIPT. Next, we analysed 269 plasma samples for the clinical validation of iSAFE NIPT. Fifty-eight of these, including five trisomies 21, two trisomies 18 and one trisomy 13 were utilised to establish the assay cut-off values based on ratios between chromosome counts. The remaining 211 plasma samples, including 10 trisomies 21, were analysed to evaluate iSAFE NIPT clinical performance.ResultsiSAFE NIPT achieved a 100% analytical sensitivity (95% CI 94.9-100% trisomy 21; 79.4-100% trisomy 18; 73.5-100% trisomy 13) and 100% specificity (95% CI 96.3-100% trisomy 21; 97.6-100% trisomy 18; 97.6-100% trisomy 13). It also achieved a 100% clinical sensitivity and specificity for trisomy 21 detection in the 211 clinical samples (95% CI for sensitivity is 69.1-100%, and 95% CI for specificity is 98.2-100%).ConclusionsThe iSAFE NIPT is a highly multiplexed ddPCR based assay for detection of fetal trisomies from maternal blood. Based on clinical validation, the iSAFE NIPT has high diagnostic sensitivity and specificity. It can be decentralized in routine clinical laboratories, is fast, easy to use and economical comparing to current NIPT.

Background NPM1 mutations have prognostic significance in acute myeloid leukemia (AML) and monitoring mutant NPM1 levels during and after therapy has been described to predict relapse and survival. Despite the published significance of this molecular biomarker, routine monitoring for mutant NPM1 levels has not been widely adopted in academic clinical laboratories. Therefore, our objective was to validate a quantitative, reverse transcription-PCR assay for the detection of NPM1 Type A mutant transcripts for use in the clinical laboratory. Methods A quantitative, real-time, reverse-transcription PCR-based method for the detection of NPM1 Type A mutant transcripts was validated for use in routine clinical practice. Results from this assay were compared to results from orthogonal methods, including next generation sequencing and digital droplet PCR. Results This real-time, reverse-transcription PCR-based method is sensitive (limit of detection: 0.0150% NCN and reproducible (≤ 0.5 log10-fold variation). We summarize the rigorous validation results and share observations that will help other clinical laboratories that may wish to implement this testing. We show the superior sensitivity of this assay compared to other assays (e.g., 45 gene Myeloid Next Generation Sequencing panel) and present a representative case which highlights the assay's utility in the pathologic assessment of cases with borderline morphologic or flow cytometric findings. Conclusions As molecular testing for residual disease in AML continues to expand, this sensitive and reproducible method will be an appropriate testing option for the detection of NPM1 Type A mutant transcripts in clinical practice.

Liquid biopsy refers to the use of various body fluids to test for circulating biological elements derived from the tumor. Liquid biopsy has taken on an increasingly important role in lung cancer diagnosis, molecular characterization, surveillance, monitoring, and determining mechanisms of resistance. These assays can utilize various sources of cell-free DNA (cfDNA) including blood, pleural fluid, urine, and others to detect tumor associated alterations. With the increasing power of next-generation sequencing technologies and the development of assays such as digital droplet PCR, rare tumor alleles can be detected in cfDNA to determine key characteristics of the tumor. Current assays, while effective, are still challenged by limited sensitivity and capacity to single genes or small panels of genes, though this is rapidly expanding. Nevertheless, testing of cfDNA has been shown to be valuable in detecting resistance to targeted inhibitors, particularly for detection of T790M in EGFR and monitoring response to therapy. With the continued development of more powerful and sensitive assays, these techniques will empower clinicians to better characterize early stage disease and can be used in the screening of high-risk patients, which may eliminate the requirement for tissue diagnosis in some settings. That said, since the majority of these alterations are not specific to lung cancer, there will continue to be a need for tissue in at least the initial diagnosis. Used in conjugation with tissue sampling, these assays will assist the treating clinician and the pathologist to better characterize individual tumors, even in the setting of limited tissue.

A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus. Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest, a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa (n = 67) and 23.5% of diseased Orbicella faveolata (n = 24). VcpA+ corals had significantly higher mortality rates and faster disease progression. For VcpA– fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata, respectively, died within 21 d of observation, while 100% of similarly sized VcpA+ fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD.

In this study, the digital droplet polymerase chain reaction (ddPCR) was used to quantify circulating bovine papillomavirus (BPV; genus: Deltapapillomavirus) DNA levels in blood samples from 25 clinically normal cows and 15 cows with chronic enzootic haematuria due to papillomavirus-associated bladder tumours. ddPCR detected BPV DNA in 95% of all the samples (i.e. in 24 of the clinically normal cows and 14 of the diseased animals), whereas quantitative real-time PCR (qPCR) detected it in only 57.5% of the same blood samples, with percentage differences between ddPCR and qPCR being statistically significant (p-value ≤ .05), according to chi-squared test. Furthermore, ddPCR detected BPV infections by a single genotype and by multiple genotypes in 37% and 63% of the cows, whereas qPCR detected these in 16% and 16%. Of the two assays, ddPCR was the more sensitive and accurate clinical diagnostic tool, allowing the detection of otherwise undetectable BPV genotypes, and consequently, a higher number of BPV co-infections. qPCR failed to detect many BPV co-infections by multiple genotypes. Therefore, ddPCR may be an essential tool for improving diagnostic procedures, allowing the identification of the genotypic distribution of BPV and a better understanding about the territorial divergence, if any, of the BPV prevalence in different areas. No significant differences in the blood viral load estimations were observed between the two animal groups, suggesting that the bloodstream could be a site of primary infection. Finally, as BPV DNA was detected in cows affected by non-invasive urothelial tumours, including papilloma and papillary urothelial neoplasms of low malignant potential, the circulating BPVs appeared to be independent of the status of urothelial neoplasms. Therefore, unlike in humans, circulating BPVs cannot be an actual prognostic marker of urothelial tumours in cows.

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.

Saliva samples were collected through a simple mouth wash procedure and viral load quantified using a technology called digital droplet PCR. Data suggest ddPCR allows for precise quantification of viral load in clinical samples infected with SARS-CoV-2.

A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus. Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest, a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa (n = 67) and 23.5% of diseased Orbicella faveolata (n = 24). VcpA+ corals had significantly higher mortality rates and faster disease progression. For VcpA– fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata, respectively, died within 21 d of observation, while 100% of similarly sized VcpA+ fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD.

Human mitochondrial DNA provides a promising target for fecal source tracking because it is unique and intrinsic to humans. We developed a TaqMan chemistry assay, hCYTB484, targeting the cytochrome b gene of the human mitochondrial genome on a droplet digital PCR (ddPCR) platform and compared the performance of hCYTB484 with the HF183/BacR287 assay, a widely used assay targeting human-associated Bacteroides. For both assays, we defined the analytical limit of detection and analytical lower limit of quantification using frequency of detection and imprecision goals, respectively. We then established these analytical limits using empirical ddPCR data, presenting a novel approach to determining the analytical lower limit of quantification. We evaluated assay sensitivity using individual human feces from US, Bangladesh, and Mozambique and evaluated assay specificity using cow, pig, chicken, and goat samples collected from the US. To compare assay performance across a range of thresholds, we utilized receiver operating characteristic curves. The hCYTB484 marker was detected and quantifiable in 100% of the human feces from the 3 geographical distant regions whereas the HF183/BacR287 marker was detectable and quantifiable in 51% and 31% (respectively) of human feces samples. The hCYTB484 marker also was more specific (97%), having fewer detections in pig, chicken, and goat samples than the HF183/BacR287 marker (80%). The higher performance of the hCYTB484 marker in individual feces from geographically distant regions is desirable in the detection of fecal pollution from sources to which fewer individuals contribute, such as the non-sewered forms of sanitation (e.g. pit latrines and septic tanks) that serve most of Earth’s population and carry the highest risk of exposure to fecal-oral pathogens., Graphical abstract Image 1, Highlights • We developed a novel TaqMan chemistry assay for the ddPCR platform targeting human mtDNA. • mtDNA was detected and quantifiable in 100% of human feces samples from the US, Mozambique, and Bangladesh. • mtDNA can complement fecal source tracking methods.

Introduction: MicroRNAs have a significant role in the pathogenesis of preeclampsia. Circulating microRNAs could represent a potential biomarker for preeclampsia. The aim of this study was to evaluate plasma miR210-3p and miR518b in preeclampsia and healthy pregnancy for the first time by digital droplet PCR (ddPCR). Methods: Thirty-six pregnant women (seventeen healthy pregnancies, nineteen preeclampsia patients) were involved from the Clinic for Gynaecology and Obstetrics “Narodni front” in Belgrade, Serbia. Plasma miR210-3p, miR518b and cel-miR-39 as a spike-in control were measured by ddPCR. Results: MiR518b was significantly elevated in preeclampsia compared to a healthy pregnancy (P = 0.034; 0.302(0.217–0.421) vs. 0.171(0.110–0.266)). MiR210-3p showed no significant difference between the two groups (P = 0.951). The adjustment of miR518b was made for a gestational age and smoking status and the difference between the preeclampsia and healthy pregnancy group was more significant (P = 0.026; 0.300(0.216–0.419) vs. 0.172(0.121–0.245)). Plasma miR-518b was significantly higher in the group of preeclampsia patients with proteinuria above the 75th percentile for the group (P=0.033), in women who smoked (P=0.039), and was positively related to uric acid in preeclampsia (P = 0.018, r = 0.536). Plasma miR518b was able to significantly discriminate between preeclampsia and healthy pregnancy, yielding AUC of 0.712 (95%CI:0.539–0.891), P = 0.028. Conclusions: In this study plasma microRNA were measured for the first time in preeclampsia and healthy pregnancies with ddPCR. Placenta-specific miR-518b could serve as a potential biomarker for discriminating preeclampsia and healthy pregnancy, which should be confirmed on a larger study population. This study has failed to confirm the same potential for miR210-3p. This article has been corrected. Link to the correction: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/3669]

// Mario Menschikowski 1 , Carsten Jandeck 1 , Markus Friedemann 1 , Brit Nacke 1 , Saskia Hantsche 1 , Oliver Tiebel 1 , Olga Sukocheva 2 and Albert Hagelgans 1 1 Institute of Clinical Chemistry and Laboratory Medicine, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany 2 School of Health Sciences, Flinders University of South Australia, Adelaide, Australia Correspondence to: Mario Menschikowski, email: Mario.Menschikowski@uniklinikum-dresden.de Keywords: liquid biopsy; free circulating DNA (fc-DNA); methylated DNA; tumor DNA; digital PCR Received: June 30, 2018 Accepted: October 24, 2018 Published: November 16, 2018 ABSTRACT Background: The analysis of aberrant DNA methylations is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique. Results: By using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl 2 concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl 2 concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers. Conclusions: We define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.

Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested., Highlights • DdPCR was used to test number of targets of validated reference assays for 4 GM crops. • At least 1 taxon-specific assay for a single DNA target was identified per crop. • Most qPCR based assays tested can be transferred to ddPCR without optimisation. • DNA digestion does not improve ddPCR characteristics at the conditions tested.

An obstacle to establishing widely useful data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. Earlier versions of EPA Methods 1609 and 1611 for enterococci used cellular reference materials for quantifying enterococci in unknown test samples, however, EPA updates to these fundamentally DNA-based analysis methods have shifted toward the use of DNA standards. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic plasmid DNA standards that have been made available for updated EPA Methods 1609.1 and 1611.1 as well as for EPA Draft Method C for Escherichia coli . To obtain the most accurate concentration estimates possible, part of this effort was to develop a data analysis model for determining the fluorescence thresholds that distinguish positive from negative droplets produced by the ddPCR reactions. Versions of this model are described for applications with individual reactions, multiple reactions within a ddPCR system run, and multiple reactions within and across different system runs. The latter version was applied toward determinations of error in the concentration estimates of the standards from replicate analyses of each standard in multiple ddPCR system runs. Mean concentration estimates for the five standards from the ddPCR analyses were 4.356, 3.381, 2.371, 1.641 and 1.071 log 10 copies/5 μL with associated standard deviations of 0.074, 0.082, 0.108, 0.131 and 0.188, respectively. These estimates contrasted with expected log10 concentrations of 4.6, 3.6, 2.6, 1.9 and 1.3 copies/5 μL, respectively, based on the yield of the plasmid reported by the vendor and spectrophotometric analysis of the initial stock solution of this material. These results illustrate how the analyses of original stocks may lead to potential bias(es) in the concentration estimates of final DNA standards and subsequently in the estimates of unknown test samples determined from these standards in qPCR analyses.

AIMS: DNA release by dying β cells leads to increased circulating levels of unmethylated preproinsulin (INS) DNA. Consistent with large-scale β cell destruction, this biomarker is elevated before and at the time of type 1 diabetes (T1D) onset. We measured serum INS DNA to evaluate whether β cells continue to undergo death in later stages of T1D. MATERIALS AND METHODS: We analyzed fasting banked sera from a cross-section of 90 participants in the T1D Exchange Registry with long standing T1D (median duration of 9 years). Subjects were determined to be C-peptide (−) or (+) based on mixed-meal tolerance testing. Results were compared to 54 adult nondiabetic controls. Stimulated samples were assayed in a subset of subjects. Levels of unmethylated and methylated INS DNA were analyzed using digital droplet PCR. RESULTS: Fasting and stimulated circulating unmethylated INS DNA levels were increased among both C-peptide (−) and C-peptide (+) subjects with longstanding T1D compared to non-diabetic controls (p

Background & Aims The accurate diagnosis of occult hepatitis B virus (HBV) infection (OBI) requires the demonstration of HBV DNA in liver biopsies of hepatitis B surface antigen-negative individuals. However, in clinical practice a latent OBI is deduced by the finding of the antibody to the hepatitis B core antigen (anti-HBc). We investigated the true prevalence of OBI and the molecular features of intrahepatic HBV in anti-HBc-positive individuals. Methods The livers of 100 transplant donors (median age 68.2 years; 64 males, 36 females) positive for anti-HBc at standard serologic testing, were examined for total HBV DNA by nested-PCR and for the HBV covalently closed circular DNA (HBV cccDNA) with an in-house droplet digital PCR assay (ddPCR) (Linearity: R2 = 0.9998; lower limit of quantitation and detection of 2.4 and 0.8 copies/105 cells, respectively). Results A total of 52% (52/100) of the individuals studied were found to have OBI. cccDNA was found in 52% (27/52) of the OBI-positive, with a median 13 copies/105 cells (95% CI 5–25). Using an assay specific for anti-HBc of IgG class, the median antibody level was significantly higher in HBV cccDNA-positive than negative donors (17.0 [7.0–39.2] vs. 5.7 [3.6–9.7] cut-off index [COI], respectively, p = 0.007). By multivariate analysis, an anti-HBc IgG value above 4.4 COI was associated with the finding of intrahepatic HBV cccDNA (odds ratio 8.516, p = 0.009); a lower value ruled out its presence with a negative predictive value of 94.6%. Conclusions With a new in-house ddPCR-based method, intrahepatic HBV cccDNA was detectable in quantifiable levels in about half of the OBI cases examined. The titer of anti-HBc IgG may be a useful surrogate to predict the risk of OBI reactivation in immunosuppressed patients. Lay summary The covalently closed circular DNA (cccDNA) form of the hepatitis B virus (HBV) sustains the persistence of the virus even decades after resolution of the symptomatic infection (occult HBV infection). In the present study we developed a highly sensitive method based on droplet digital PCR technology for the detection and quantitation of HBV cccDNA in the liver of individuals with occult HBV infection. We observed that the amount of HBV cccDNA may be inferred from the titer in serum of the IgG class antibody to the hepatitis B core antigen. The quantitation of this antibody may represent a surrogate to determine which patients are at the highest risk of HBV reactivation following immunosuppressive therapies.

Background JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden. Methods Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed. Results Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P

Accurate food labelling is of utmost importance for food safety and consumer choice in the food chain. Complete or partial substitution, whether intentional or unintentional, may introduce food pathogens or allergens to a product or affect personal or religious beliefs. Several studies around the world have reported different degrees of species substitution in meat products but no similar studies have been conducted in the Canadian market for sausage products. In this study, 100 raw meat sausage samples that were labelled as single meat species products (beef, pork, chicken or turkey) were collected from retail establishments across Canada and were surveyed for the presence of a panel of non-labeled species. The predominant meat species were determined using DNA barcoding and contaminant or unclaimed meat species were detected using digital droplet PCR using species specific primers and probes. All samples were also tested for presence of horse meat using real-time PCR. All samples contained the predominant species matching the label species except for five turkey sausage samples which contained chicken as the predominant species. Second, this analysis showed that 6% of beef sausages also contained pork, 20% of chicken sausages contained turkey while 5% contained beef, and 5% of pork sausages also contained beef. Five samples labeled as turkey sausage contained no turkey and one pork sample was found to contain horse meat. The overall mislabeling rate detected in this study was 20% and the results provide a baseline for assessing species mislabeling in processed meat products in Canada.

// Pieter A. Boonstra 1 , Arja ter Elst 2 , Marco Tibbesma 1, 2 , Lisette J. Bosman 2 , Ron Mathijssen 3 , Florence Atrafi 3 , Frits van Coevorden 4 , Neeltje Steeghs 5 , Sheima Farag 5 , Hans Gelderblom 6 , Winette T.A. van der Graaf 7 , Ingrid M.E. Desar 7 , Jacqueline Maier 8 , Jelle Overbosch 9 , Albert J.H. Suurmeijer 2 , Jourik Gietema 1 , Ed Schuuring 2, * and Anna K.L. Reyners 1, * 1 University of Groningen, University Medical Center Groningen, Department of Medical Oncology, Groningen 9713 GZ, The Netherlands 2 University of Groningen, University Medical Center Groningen, Department of Pathology, Groningen 9713 GZ, The Netherlands 3 Department of Medical Oncology, Erasmus University Medical Center Rotterdam, Rotterdam 3015 CE, The Netherlands 4 Antoni van Leeuwenhoek, Netherlands Cancer Institute, Department of Surgery, Amsterdam 1066 CX, The Netherlands 5 Antoni van Leeuwenhoek, Netherlands Cancer Institute, Department of Medical Oncology, Amsterdam 1066 CX, The Netherlands 6 Leiden University Medical Center, Department of Medical Oncology, Leiden 2300 RC, The Netherlands 7 Radboud University Medical Center, Department of Medical Oncology, Nijmegen 6500 HB, The Netherlands 8 University of Leipzig, Center for Internal Medicine, Department of Hematology/Oncology, Leipzig 04103, Germany 9 University of Groningen, University Medical Center Groningen, Department of Radiology, Groningen 9713 GZ, The Netherlands * These authors share authorship Correspondence to: Anna K.L. Reyners, email: a.k.l.reyners@umcg.nl Keywords: GIST; cell-free circulating DNA; single assay; plasma; digital droplet PCR Received: October 05, 2017 Accepted: January 13, 2018 Published: February 14, 2018 ABSTRACT Background: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients’ plasma. Methods: A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample. Results: The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease. Conclusion: A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

Under the current pandemic situation caused by the novel coronavirus SARS-CoV-2, wastewater monitoring has been increasingly investigated as a surveillance tool for community-wide disease prevalence. After a year into the pandemic, this review critically discusses the real progress made in the detection of SARS-CoV-2 using wastewater monitoring. The limitations and the key challenges faced in improving the detection methods are highlighted. As per the literature, the complex nature of the wastewater matrix poses problems in processing the samples and achieving high sensitivity at low loads of viral RNA using the current detection methods. Furthermore, in the absence of a gold standard analytical method for wastewater, the validation of the generated data for use in wastewater-based epidemiological modeling of the disease becomes practically difficult. However, research is advancing in adopting clinical methods to the wastewater by using appropriate processing controls, and recovery methods. Besides, the technological advances made by the industry including the development of PCR kits with improved detection limits, easy-to-use viral RNA concentration methods, ability to detect the coronavirus variants, and artificial intelligence and advanced data modeling for continuous and remote monitoring greatly help to debottleneck some of these problems. Currently, these technologies are limited to healthcare systems, however, their use for wastewater monitoring is expected to provide opportunities for wide-scale applications of wastewater-based epidemiology (WBE). Moreover, the data from wastewater monitoring act as the initial checkpoint for human health even before the appearance of symptoms, hence WBE needs more attention to manage current and future infectious transmissions., Graphical Abstract ga1

The cobas® EGFR Test provides a semiquantitative index (SQI) that reflects the proportion of mutated versus wild-type copies of the EGFR gene in plasma. The significance of SQI as an indirect measure of the variant allele frequency (VAF) or mutated copies/mL remains unclear. The aim of this study was to evaluate the correlation of SQI with the VAF and the number of mutated copies/mL obtained by a digital droplet PCR (ddPCR) test in NSCLC samples. The study included 118 plasma samples from a retrospective cohort of 25 stage IV adenocarcinoma patients with EGFR exon 19 deletions (Ex19Del), obtained before and during tyrosine kinase inhibitor (TKI) treatment. Both SQI and VAF and SQI and mutated copies/mL showed the same significant correlation (r2 = 0.79, p &lt, 0.00001) across the whole study cohort. We found better correlation in samples collected at the baseline between SQI and VAF (r2 = 0.94, p &lt, 0.00001) and SQI and mutated copies/mL (r2 = 0.97, p &lt, 0.00001) compared to samples collected during TKI treatment: r2 = 0.76, p &lt, 0.00001 for SQI and VAF and r2 = 0.75, 0.00001 for SQI and mutated copies/mL. The study indicates that SQI is a robust quantitative indirect measure of VAF and the number of mutated copies/mL in plasma from patients with an EGFR Ex19Del mutation. Further studies are desirable to assess the SQI cut-off values related to the clinical status of the patient.

The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (

Purpose Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR). Methods Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR. Droplets positive for VIC and FAM fluorescence were read in a QX200 Droplet reader™ (BIO-RAD) after which the viral load was calculated using Quantasoft software. Results We found that DNAs extracted from formalin fixed paraffin embedded (FFPE) tissue samples yielded lower amplification signals compared to those obtained from liquid based cytology (LBC) samples, but they were clearly distinguishable from negative background signals. The viral limit of detection was 1.6 copies of HPV 16, 2.8 copies of HPV 18, 4.6 copies of HPV 33 and 1.6 copies of HPV 45. The mean inter-assay coefficients of variability (CV) for the assays ranged from 3.4 to 7.0%, and the mean intra-assay CV from 2.6 to 8.2%. The viral load in the different cohorts of tumor samples ranged from 154 to 340,200 copies for HPV 16, 244 to 31,300 copies for HPV 18 and 738 to 69,100 copies for HPV 33. One sample positive for HPV 45 contained 1331 viral copies. When comparing qPCR data with ddPCR copy number data, the qPCR values were found to be 1 to 31 times higher. Conclusions Separation of fragments in nanodroplets may facilitate the amplification of fragmented human and viral DNA. The method of digital droplet PCR may, thus, provide a new and promising tool for evaluating the HPV viral load in clinical samples.