Positive Droplets Research Articles

Getting rid of 'rain' and 'stars': Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg.

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets ('stars'). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets ('stars') and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).

Effect of SARS-CoV-2 digital droplet RT-PCR assay sensitivity on COVID-19 wastewater based epidemiology

We developed and implemented a framework for examining how molecular assay sensitivity for a viral RNA genome target affects its utility for wastewater-based epidemiology. We applied this framework to digital droplet RT-PCR measurements of SARS-CoV-2 and Pepper Mild Mottle Virus genes in wastewater. Measurements were made using 10 replicate wells which allowed for high assay sensitivity, and therefore enabled detection of SARS-CoV-2 RNA even when COVID-19 incidence rates were relatively low (~10−5). We then used a computational downsampling approach to determine how using fewer replicate wells to measure the wastewater concentration reduced assay sensitivity and how the resultant reduction affected the ability to detect SARS-CoV-2 RNA at various COVID-19 incidence rates. When percent of positive droplets was between 0.024% and 0.5% (as was the case for SARS-CoV-2 genes during the Delta surge), measurements obtained with 3 or more wells were similar to those obtained using 10. When percent of positive droplets was less than 0.024% (as was the case prior to the Delta surge), then 6 or more wells were needed to obtain similar results as those obtained using 10 wells. When COVID-19 incidence rate is low (~ 10−5), as it was before the Delta surge and SARS-CoV-2 gene concentrations are <104 cp/g, using 6 wells will yield a detectable concentration 90% of the time. Overall, results support an adaptive approach where assay sensitivity is increased by running 6 or more wells during periods of low SARS-CoV-2 gene concentrations, and 3 or more wells during periods of high SARS-CoV-2 gene concentrations.

Image Segmentation and Quantification of Droplet dPCR Based on Thermal Bubble Printing Technology.

Thermal inkjet printing can generate more than 300,000 droplets of picoliter scale within one second stably, and the image analysis workflow is used to quantify the positive and negative values of the droplets. In this paper, the SimpleBlobDetector detection algorithm is used to identify and localize droplets with a volume of 24 pL in bright field images and suppress bright spots and scratches when performing droplet location identification. The polynomial surface fitting of the pixel grayscale value of the fluorescence channel image can effectively compensate and correct the image vignetting caused by the optical path, and the compensated fluorescence image can accurately classify positive and negative droplets by the k-means clustering algorithm. 20 µL of the sample solution in the result reading chip can produce more than 100,000 effective droplets. The effective droplet identification correct rate of 20 images of random statistical samples can reach more than 99% and the classification accuracy of positive and negative droplets can reach more than 98% on average. This paper overcomes the problem of effectively classifying positive and negative droplets caused by the poor image quality of photographed picolitre ddPCR droplets caused by optical hardware limitations.

Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries

Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15–145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.

Application of a novel fluorogenic polyurethane analogue probe in polyester-degrading microorganisms screening by microfluidic droplet.

Application of polyester-degrading microorganisms or enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. However, current impranil DLN-based screening of polyester-degrading microorganisms is time-consuming, labour-intensive and unable to distinguish polyesterases from other protease- or amidase-like enzymes. Herein, we present an approach that combined a novel synthetic fluorescent polyurethane analogue probe (FPAP), along with the droplet-based microfluidics to screen polyurethane-degrading microorganisms through fluorescence-activated droplet sorting (FADS) pipeline. The fluorescent probe FPAP exhibited a fluorescence enhancement effect once hydrolysed by polyesterases, along with a strong specificity in discriminating polyesterases from other non-active enzymes. Application of FPAP in a microfluidic droplet system demonstrated that this probe exhibited high sensitivity and efficiency in selecting positive droplets containing leaf-branch compost cutinase (LCC) enzymes. This novel fluorogenic probe, FPAP, combined with the droplet microfluidic system has the potential to be used in the exploitation of novel PUR-biocatalysts for biotechnological and environmental applications.

Endovascular Biopsy for Detection of Somatic Mosaicism in Human Fusiform Cerebral Aneurysms

Endovascular Biopsy for Detection of Somatic Mosaicism in Human Fusiform Cerebral Aneurysms

Time Course of Fat Accumulation in Various Organs of Young OLETF Rats

PurposeExcessive fat accumulation induces chronic inflammation in the white adipose tissue (WAT), which leads and accelerates to obesity‐related health problems such as glucose intolerance, insulin resistance, and dyslipidemia. Determination of the time course of fat accumulation in obesity might allow identification of target tissues to treat the health problems in each stage. The purpose of the present study was to investigate the relationship between inflammatory changes and time course of fat accumulation in various organs.MethodsMale Otsuka Long‐Evans Tokushima fatty (OLETF) rats were used as spontaneous obesity animals. All rats were fed a standard diet containing 55.3% carbohydrate and water ad libitum. From 4‐ to 20‐week‐old, blood samples from vena cava and samples of the epididymal adipose tissue, retroperitoneal adipose tissue, mesenteric adipose tissue, and inguinal subcutaneous adipose tissue as WAT, brown adipose tissue (BAT) from interscapular region, liver, pancreas, and plantaris muscles were analyzed histologically every 2 weeks. Collected serum was used to detect triglyceride (TG) and free fatty acid (FFA) concentrations. Tissue samples were sliced and stained with hematoxylin and eosin or oil red O for histological observation.ResultsBody weight in OLETF rats increased linearly from 4‐ to 20‐week‐old. Although the wet weight and adipocyte diameter of the epididymal adipose tissue also increased throughout the experimental period, the values of inguinal subcutaneous adipose tissue increased by 10‐week‐old and were almost the same after 10‐week‐old. The liver TG levels increased from 4‐ to 8‐week‐old. However, hypertriglyceridemia, BAT whitening, and hepatic steatosis were not observed at this period. The serum TG levels increased after 10‐week‐old. Additionally, the number of whitened unilocular adipocytes with a diameter &gt;10 μm in the BAT increased after 10‐week‐old and oil red O positive lipid droplets were usually observed in the liver at this period. Not only the serum TG levels but also the serum FFA levels increased after 18‐week‐old. Additionally, enlarged adipocytes with a diameter &gt;130 μm and crown like structure indicating inflammatory change were often observed in the epididymal adipose tissue and retroperitoneal adipose tissue, not in mesenteric adipose tissue and inguinal subcutaneous adipose tissue, at this period. Fatty pancreas and intramuscular fat deposition were not observed throughout the experimental period.ConclusionAdipocyte hypertrophy in the visceral WAT and fatty liver could precede dyslipidemia in young OLETF rats and continuous obesity subsequently proceed to ectopic fat accumulation including BAT whitening and chronic inflammation in the epididymal adipose tissue and retroperitoneal adipose tissue with higher FFA levels. Hyperphagia mainly on carbohydrate could contribute to ectopic fat accumulation in the liver and BAT whitening, not to fatty pancreas and intramuscular fat deposition in young stage.

Using Droplet Digital PCR to Detect Cyanobacteria in Human Lung Tissue

Objective and HypothesisCyanobacteria produce a variety of secondary metabolites, including toxins, that are harmful to human and animal health. The objective of this study is to develop a quantitative method that can reliably detect cyanobacteria in human lung tissue. We propose that with cyanobacteria‐specific 16S rDNA primers, droplet digital PCR (ddPCR) can provide a reliable and sensitive method to detect Cyanobacteria in human tissue.MethodsTwo cyanobacteria strains were used in this validation: a toxin‐producing strain, Microcystis aeruginosa (LB 2385), and a non‐toxin‐producing, strain Synechococcus sp. (LB 2390). Escherichia coli served as a negative bacterial control. DNA was extracted from cultures and frozen, human lung tissue with DNeasy Blood &amp; Tissue kit (QIAGEN) and quantified using the dsDNA High‐Sensitivity Assay (Invitrogen) on a Qubit 3.0 fluorometer. The ddPCR reactions consisted of FAM labelled cyanobacterial 16S rDNA target primer/probe and HEX labelled RPP30 reference primer/probe (Bio‐RAD). Oil droplets were generated using the Bio‐Rad Automated Droplet Generator and the emulsified PCRs were run in a 96‐well plate on a C1000 Touch™ Thermal Cycler. The plates were read on a Bio‐Rad QX200 droplet reader and the data analyzed using the QuantaSoft v1.7.4 software. The Limit of Detection (LOD) was found by initially diluting LB 2385 and LB 2390 to 4 ng/mcL in nuclease‐free water, followed by serial 2x dilutions until it was below the detection limit by ddPCR. This was repeated using normal human lung tissue instead of nuclease‐free water.ResultsThe ddPCR assay developed in this study showed high specificity and sensitivity in detecting both strains of cyanobacteria. Under both conditions tested (spiking in water vs 10 ng human DNA (hDNA)), LB 2385 DNA could be reliably detected at a concentration of 61 fg/mcL, whereas LB 2390 DNA could be reliably detected at a concentration of 31 fg/mcL. This corresponds to 1‐2 copies/mcL of cyanobacterial DNA in the original samples. In the presence of hDNA, the sensitivity for cyanobacterial DNA decreased as the concentration of hDNA increased (2‐30 ng/mcL), possibly due to a physical barrier or competition between the primer/probes. In the hDNA condition, RPP30 could be reliably detected at 2‐3 copies/mcL with a concentration of ~60 pg/mcL. RPP30 is valuable for normalizing sample input, reducing biases caused by DNA quantification variations and pipetting errors. A strong linear correlation between sample input and the number of positive droplets (i.e., the concentration detected by ddPCR) was observed for both cyanobacteria 16S rDNA and RPP30 DNA targets. This assay also demonstrated high intra‐ and inter‐assay reproducibility.ConclusionsA highly specific and sensitive ddPCR assay was developed to detect the presence of cyanobacteria in human lung tissue. The ability to detect cyanobacteria in human samples will allow further research into the role cyanobacteria plays in human health and disease.

A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR.

Simple SummaryDroplet digital polymerase chain reaction (ddPCR) is an innovative technique for quantifying a target DNA in a diluted target sample, based on the partition of PCR reaction in a large number of sub-reactions (droplets). In recent years there has been an increase in digital PCR (dPCR) utilization in several different fields, and the application of ddPCR in fungicide resistance studies is very recent. Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB), one of the most devasting foliar diseases of wheat grown in temperate climates. STB control relies mostly on fungicide applications and mutations conferring fungicides resistances are concerning phenomena. G143A substitution in fungal cytochrome bc1 confers resistance toward Quinone outside Inhibitor (QoIs) fungicides. In Italy, QoIs are currently sprayed in STB control programs. To the best of our knowledge, we have developed the first ddPCR assay for G143 and A143 alleles detection in samples of gDNA from Z. tritici monocondial cultures. We have also investigated G143 and A143 alleles frequency in Italian Z. tritici populations representative of different fungicide management strategies. The detection of very low G143A substitution percentages in Z. tritici populations is essential for monitoring the emergence of QoIs resistance in field and effectively control STB.Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins’ efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease.

Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics

SummaryWe report a droplet microfluidic method to target and sort individual cells directly from complex microbiome samples and to prepare these cells for bulk whole-genome sequencing without cultivation. We characterize this approach by recovering bacteria spiked into human stool samples at a ratio as low as 1:250 and by successfully enriching endogenous Bacteroides vulgatus to the level required for de novo assembly of high-quality genomes. Although microbiome strains are increasingly demanded for biomedical applications, a vast majority of species and strains are uncultivated and without reference genomes. We address this shortcoming by encapsulating complex microbiome samples directly into microfluidic droplets and amplifying a target-specific genomic fragment using a custom molecular TaqMan probe. We separate those positive droplets by droplet sorting, selectively enriching single target strain cells. Finally, we present a protocol to purify the genomic DNA while specifically removing amplicons and cell debris for high-quality genome sequencing.

The Effect of Cytomegalovirus on Pediatric Acute Lymphoblastic Leukemia

Background: Recent evidence supports the role of cytomegalovirus (CMV) in the development of childhood ALL. The underlying mechanism and CMV's role in the leukemic cell phenotype is unknown, but CMV typically interacts with the host immune system allowing the virus to survive in a latent state; it may be that this immune dysregulation affects the risk of ALL. This study aims to explore the association of CMV and ALL at the time of leukemia diagnosis, using AML cases as a control and further, to determine whether CMV affects certain subgroups of ALL patients such as specific ethnicities, age groups, or cytogenetic subtypes.Methods: Pediatric diagnostic leukemia bone marrow samples obtained from the California Childhood Leukemia Study were screened for the presence of CMV DNA using a custom-designed droplet digital PCR assay. A total of 869 cases were analyzed including 125 AML cases and 744 ALL cases. Demographic and clinical features were compared between patients found to be CMV positive (cases with any detectable CMV positive droplets) and those who were CMV negative (cases with no detectable CMV positive droplets). The effect of the level of CMV viral DNA load was also assessed. For a subset of cases (n=61), Affymetrix Array gene expression data were available and differential gene expression performed to compare CMV positive cases with high viral load to CMV negative cases. Odds ratios and confidence intervals were estimated using logistic regression.Results: ALL cases were more likely to be CMV positive compared to AML (OR: 2.50; CI 1.00, 5.47, p = 0.039 for CMV highest quintile vs. CMV negative). Within ALL cases, B-cell ALL (B-ALL) was significantly associated with CMV positivity compared to T-cell ALL (T-ALL) (OR: 2.93; CI: 1.01, 8.52, p = 0.048 for CMV highest tertile vs. CMV negative). Further subtype analysis of B-ALL cases revealed CMV positivity to be significantly associated with high hyperdiploidy, one of the most common ALL subtypes, when compared to ETV6-RUNX1 (OR: 2.52; CI:1.34, 4.73, p = 0.004 for highest CMV tertile vs. CMV negative). CMV positive B-ALL cases were also more likely to harbor deletions of EBF1, a B-cell development gene, compared to CMV negative cases (OR: 6.10; CI: 1.09, 34.06, p = 0.04 for CMV 2 nd tertile vs. CMV negative; OR: 5.54; CI: 1.13, 27.18, p = 0.03 for CMV highest tertile vs. CMV negative). Differential gene expression analysis revealed 830 genes to be significantly differentially expressed between the highest quintile of CMV positive cases and CMV negative cases, and gene ontology analysis revealed upregulation of processes involved in viral infection and replication. Specifically, cytokine signaling pathways including IL-1, IL-8, and IL-7 were upregulated in CMV positive cases while Th1 and the pathway facilitating crosstalk between dendritic cells and natural killer cells were downregulated. Interestingly, B-cell receptor signaling was also upregulated in CMV positive cases.Conclusion: Our results support the hypothesis that CMV plays an enhanced role in leukemia development in specific subtypes of ALL, and not in AML development. The ability of CMV to interact with the host immune system, highlights immune dysregulation as a potential mechanism by which CMV contributes to risk of ALL. Gene expression analysis on a subset of cases revealed differentially expressed genes to be enriched in pathways involved in immune response, suggesting a potential role for active CMV infection in the leukemic phenotype. The patterns of up- and downregulation in these pathways were consistent with the host response to CMV. Additionally, acute CMV infection has been shown to promote B-cell activation and proliferation. Further, CMV seropositive individuals have been reported to have altered immune responses even with the virus in a latent state highlighting the virus's effect on B-cells. In our study we also found B-cell receptor signaling to be upregulated in CMV positive cases. This is consistent with the known effects of CMV in a typical host, but in patients with leukemia it provides an interesting potential link between CMV infection and development of pediatric ALL. These intriguing results require validation and warrant continued investigation of the role of CMV in pediatric leukemia development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MYOD1 c.365G&amp;gt;T, p.L122R Variant Detection by Droplet Digital PCR (ddPCR)

Abstract Introduction/Objective Rhabomyosarcomas (RMS) are a group of skeletal muscle tumors that include embryonal, alveolar, pleomorphic, spindle cell/sclerosing subtypes (SC/SRMS). Spindle cell RMS occurs in both adult and pediatric populations, and is associated with either more aggressive or better clinical outcomes respectively. A recurrent hotspot variant in MYOD1, p.L122R (NM_002478.4 c.365G&amp;gt;T), has been described in SC/SRMS. The classification of this diagnosis is evolving, with VGLL2 and NCOA2 fusions defining the diagnosis in young children, and MYOD1 p.L122R defining the diagnosis in older children. The MYOD1 p.L122R variant seems to be associated with more aggressive disease, and may be increasingly used in risk stratification with intensification of treatment. Methods/Case Report A digital droplet PCR (ddPCR) assay was used to detect the MYOD1 p.L122R in DNA samples with RMS. Patients and controls were coded as positive or negative, and tested for association with clinical features and outcome. Results (if a Case Study enter NA) Known-positive cohort of samples was limited by the extreme rarity of this tumor. “Known-positive” status was established by confirmation of the variant with an external clinically-validated assay. The six known positive samples were assessed by ddPCR for the presence of MYOD1 L122R. The L122R variant was detected in all six variants for a sensitivity of 100%. DNA and/or TNA obtained from known wild-type FFPE and frozen material was assessed, for a total of nine unique samples (1 synthetic, 8 patient-derived). All 9 samples were wild- type, with no positive droplets detected, for a specificity of 100%. Conclusion Our MYOD1 c.365G&amp;gt;T, p.L122R variant detection by droplet digital PCR (ddPCR) assay is a robust, reproducible, specific and sensitive method to detect the MYOD1 hotspot mutation.

42P Method optimization for the detection of chimerism by real-time PCR and droplet digital PCR

The objective of the present study was to select combinations of primers for genetic polymorphisms (INDELS) used in the analysis of chimerism associated with post-transplant hematopoietic regeneration as well as to test them under real-time PCR (qPCR) and droplet digital PCR (ddPCR) conditions. We searched for genetic polymorphisms, using the EMBL - European Molecular Biology Laboratory - database taking into account certain criteria for selection. There were 85 polymorphisms left at the end of the selection process. For both techniques, we designed primers with the following characteristics: 65°C annealing temperature, amplicon length below 120 bp, at least 3 different nucleotides at the 3’ end between variants and temperature difference between forward and reverse primer of maximum 1°C. Depending on the distribution of genotypes, assays were designed for one or both alleles. Human blood samples from subjects without hematological diseases were obtained in accordance with the Helsinki declaration and genotyped. A novel genotyping protocol, based on 2 reference genes and one allele per polymorphism, was developed. Samples bearing different genotypes were serially diluted to simulate various chimerism levels. In general, for ddPCR increasing primer concentration determines the increase of the amplitude difference between the negative and the positive droplets without affecting the allele specificity. At the same time, the decrease of the annealing extension temperature determined the same phenomenon. Some of the assays showed no differences between 35 and 45 cycles. The new protocol allows the use of smaller sample volumes as compared to traditional genotyping, by reducing reaction number and template quantity. Simulated and obtained chimerism levels showed good correlation between 50% and 1.5% (R2=0.998), the limit of quantification corresponding to 50 molecules. The differences between positive and negative samples are more clearly determined in the case of ddPCR, which confirms that this method is more accurate than the qPCR method. In this context we believe that the ddPCR is a valuable technique for early detection in hematopoietic malignancies. Ioana M. Lambrescu and Victor S. Ionescu - equal contribution.

Droplet digital PCR as a sensitive tool to assess exposure pressure from Echinococcus multilocularis in intermediate hosts

A key element to understanding parasite epidemiology is assessing their prevalence in the respective wild reservoir hosts. The tapeworm Echinococcus multilocularis circulates between canid species (definite hosts) and small mammals (mostly rodents; intermediate hosts). Prevalence rates of Echinococcus multilocularis in the intermediate host are most exclusively determined through macroscopic examination of the liver generally followed by molecular or histological diagnostic for parasite species confirmation. The overall objective of the study was to investigate the suitability of Real-Time PCR and Droplet Digital PCR (ddPCR) analysis as tool to detect exposure pressure (frequency of infection events) from E. multilocularis in intermediate hosts even in the absence of macroscopic lesions in the liver. One hundred six small mammals (meadow voles and deer mice) were trapped followed by post-mortem examination including macroscopic evaluation of the liver to detect lesions indicative of infection with Echinococcus multilocularis but also by sampling a piece of liver in absence of lesion to submit it to molecular assay. Macroscopic lesions were present in the livers of two samples. Including the latter two samples, five samples yielded a positive result following Real-Time PCR, whereas 16 samples displayed three or more positive droplets upon ddPCR and were considered positive. Whether these additional cases without macroscopic lesions would have become infectious during the lifespan of the rodent or were abortive or early infections is unclear, but these data suggest levels of exposure of intermediate hosts to the parasite is much higher than assumed.

Development and Evaluation of a Droplet Digital PCR Assay for 8p23 β-Defensin Cluster Copy Number Determination.

IntroductionIt has been proposed that the copy number (CN) variation (CNV) in β-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available.ObjectiveDroplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay.MethodsThe assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes.ResultsIn a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy.ConclusionsddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case–control studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00546-2.

Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients

BackgroundMutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC.MethodsPrimers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR.ResultsUsing methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level.ConclusionsOur study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

Periodontal Pathogens Promote Foam Cell Formation by Blocking Lipid Efflux

Foam cells are one of the major cellular components of atherosclerotic plaques, within which the trace of periodontal pathogens has also been identified in recent studies. In line with these findings, the correlation between periodontitis and atherosclerotic cardiovascular incidences has been repetitively supported by evidence from a number of experimental studies. However, the direct role of periodontal pathogens in altered cellular signaling underlying such cardiovascular events has not been clearly defined. To determine the role of periodontal pathogens in the pathogenesis of atherosclerosis, especially in the evolution of macrophages into foam cells, we monitored the pattern of lipid accumulation within macrophages in the presence of periodontal pathogens, followed by characterization of these lipids and investigation of major molecules involved in lipid homeostasis. The cells were stained with the lipophilic fluorescent dye BODIPY 493/503 and Oil Red O to characterize the lipid profile. The amounts of Oil Red O–positive droplets, representing neutral lipids, as well as fluorescent lipid aggregates were prominently increased in periodontal pathogen–infected macrophages. Subsequent analysis allowed us to locate the accumulated lipids in the endoplasmic reticulum. In addition, the levels of cholesteryl ester in periodontal pathogen–infected macrophages were increased, implying disrupted lipid homeostasis. Further investigations to delineate the key messengers and regulatory factors involved in the altered lipid homeostasis have revealed alterations in cholesterol efflux–related enzymes, such as ABCG1 and CYP46A1, as contributors to foam cell formation, and increased Ca2+ signaling and reactive oxygen species (ROS) production as key events underlying disrupted lipid homeostasis. Consistently, a treatment of periodontal pathogen–infected macrophages with ROS inhibitors and nifedipine attenuated the accumulation of lipid droplets, further confirming periodontal pathogen–induced alterations in Ca2+ and ROS signaling and the subsequent dysregulation of lipid homeostasis as key regulatory events underlying the evolution of macrophages into foam cells.

Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load.

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (<5 min) generation of several thousand monodispersed droplets ∼150 to 350 μm in size with a CV of ∼2.3%. A block copolymer surfactant (polyoxyalkylene F127) is used to stabilize these large droplets in oil during thermal cycling. At this droplet size and number, positive droplets can be counted by eye or imaged by a smartphone with appropriate illumination/filtering to accurately quantify up to 100 target copies. We demonstrate with 2019 nCoV-PCR assay LODs of 3.8 copies per 20 μL of sample and a dynamic range of 4-100 copies. The ddPCR platform is shown to be inhibitor resistant with spiked saliva samples, suggesting RNA extraction may not be necessary. It represents a rapid 1.5-h POC quantitative PCR test that requires just a pipette equipped with elliptical pipette tip, a commercial portable thermal cycler, a smartphone, and a portable trans-illuminator, without bulky and expensive micropumps and optical detectors that prevent POC application.

Aerosol and Droplet Risk of Common Otolaryngology Clinic Procedures.

Define aerosol and droplet risks associated with routine otolaryngology clinic procedures during the COVID-19 era. Clinical procedures were simulated in cadaveric heads whose oral and nasal cavities were coated with fluorescent tracer (vitamin B2) and breathing was manually simulated through retrograde intubation. A cascade impactor placed adjacent to the nares collected generated particles with aerodynamic diameters ≤14.1 µm. The 3D printed models and syringes were used to simulate middle and external ear suctioning as well as open suctioning, respectively. Provider's personal protective equipment (PPE) and procedural field contamination were also recorded for all trials using vitamin B2 fluorescent tracer. The positive controls of nebulized vitamin B2 produced aerosol particles ≤3.30 µm and endonasal drilling of a 3D model generated particles ≤14.1 µm. As compared with positive controls, aerosols and small droplets with aerodynamic diameter ≤14.1 µm were not detected during rigid nasal endoscopy, flexible fiberoptic laryngoscopy, and rigid nasal suction of cadavers with simulated breathing. There was minimal to no field contamination in all 3 scenarios. Middle and external ear suctioning and open container suctioning did not result in any detectable droplet contamination. The clinic suction unit contained all fluorescent material without surrounding environmental contamination. While patients' coughing and sneezing may create a baseline risk for providers, this study demonstrates that nasal endoscopy, flexible laryngoscopy, and suctioning inherently do not pose an additional risk in terms of aerosol and small droplet generation. An overarching generalization cannot be made about endoscopy or suctioning being an aerosol generating procedure. 3.

Getting rid of rain and stars: mitigating inhibition effects in ddPCR assays, the case of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg

ddPCR is getting more and more popular in the field of eDNA-based aquatic monitoring. Even if emulsion PCR used in ddPCR confers a partial resistance to inhibition due to the high number of reactions for the same sample (between 10K and 20K), it is not impervious to it. Inhibition impacts the fluorescence amplitude of positive droplets, affecting both their dispersion and their position relatively to the negative droplets cloud. This fluctuation could jeopardize the use of a shared threshold among several samples and thus the objective assignation of the positive droplets. This is even more critical for low concentration samples such as eDNA samples: the positive droplets are scarce and it is thus crucial to objectively discriminate if they can be counted as positive by establishing an appropriate threshold. Another issue is the artifactual generation of high fluorescence droplets that could be counted as positive with a single threshold solution. Here we propose a double threshold method to take both high fluorescence droplets and PCR inhibition impact into account allowing for an objective sorting of the positive and negative droplets in ddPCR assays.