The Next Frontier in Noninvasive Prenatal Diagnostics: Cell-Free Fetal DNA Analysis for Monogenic Disease Assessment.

With the widespread clinical adoption of noninvasive screening for fetal chromosomal aneuploidies based on cell-free DNA analysis from maternal plasma, more researchers are turning their attention to noninvasive prenatal assessment for single-gene disorders. The development of a spectrum of approaches to analyze cell-free DNA in maternal circulation, including relative mutation dosage, relative haplotype dosage, and size-based methods, has expanded the scope of noninvasive prenatal testing to sex-linked and autosomal recessive disorders. Cell-free fetal DNA analysis for several of the more prevalent single-gene disorders has recently been introduced into clinical service. This article reviews the analytical approaches currently available and discusses the extent of the clinical implementation of noninvasive prenatal testing for single-gene disorders.

Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay.

While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

IMPORTANCE: Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.

Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Mitochondrial DNA in bronchoalveolar lavage fluid is associated with the prognosis of idiopathic pulmonary fibrosis: a single cohort study.

BACKGROUND: Extracellular mitochondrial DNA (mtDNA) is released from damaged cells and increases in the serum and bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) patients. While increased levels of serum mtDNA have been reported to be linked to disease progression and the future development of acute exacerbation (AE) of IPF (AE-IPF), the clinical significance of mtDNA in BALF (BALF-mtDNA) remains unclear. We investigated the relationships between BALF-mtDNA levels and other clinical variables and prognosis in IPF. METHODS: Extracellular mtDNA levels in BALF samples collected from IPF patients were determined using droplet-digital PCR. Levels of extracellular nucleolar DNA in BALF (BALF-nucDNA) were also determined as a marker for simple cell collapse. Patient characteristics and survival information were retrospectively reviewed. RESULTS: mtDNA levels in serum and BALF did not correlate with each other. In 27 patients with paired BALF samples obtained in a stable state and at the time of AE diagnosis, BALF-mtDNA levels were significantly increased at the time of AE. Elevated BALF-mtDNA levels were associated with inflammation or disordered pulmonary function in a stable state (n = 90), while being associated with age and BALF-neutrophils at the time of AE (n = 38). BALF-mtDNA ≥ 4234.3 copies/µL in a stable state (median survival time (MST): 42.4 vs. 79.6 months, p < 0.001) and ≥ 11,194.3 copies/µL at the time of AE (MST: 2.6 vs. 20.0 months, p = 0.03) were associated with shorter survival after BALF collection, even after adjusting for other known prognostic factors. On the other hand, BALF-nucDNA showed different trends in correlation with other clinical variables and did not show any significant association with survival time. CONCLUSIONS: Elevated BALF-mtDNA was associated with a poor prognosis in both IPF and AE-IPF. Of note, at the time of AE, it sharply distinguished survivors from non-survivors. Given the trends shown by analyses for BALF-nucDNA, the elevation of BALF-mtDNA might not simply reflect the impact of cell collapse. Further studies are required to explore the underlying mechanisms and clinical applications of BALF-mtDNA in IPF.

Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells.

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional ß-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). METHODS: After HSPCs were transduced with an LV encoding the therapeutic ß-globin (ßA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. RESULTS: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of ßA-T87Q protein detected by reverse-phase high-performance liquid chromatography. CONCLUSIONS: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

Analysis of three characterization assays reveals ddPCR of LIN28A as the most sensitive for the detection of residual pluripotent stem cells in cellular therapy products.

BACKGROUND AIMS: With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. DISCUSSION: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.

Revealing parental mosaicism: the hidden answer to the recurrence of apparent de novo variants.

Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (EYA1 and EBF3) and one family has paternal gonadal mosaicism (CHD7) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (FLNC) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7-35.9%, limit of detection 0.08-0.16% based on our in-house EBF3 assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.

Detection and quantification of JAK2V617F copy number by droplet digital PCR versus real-time PCR.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.

Clinical evaluation of a multiplex droplet digital PCR for pathogen detection in critically ill COVID-19 patients with bloodstream infections.

BACKGROUND: Nosocomial bloodstream infections (nBSI) have emerged as a clinical concern for physicians treating COVID-19 patients. In this study, we aimed to evaluate the effectiveness of a multiplex ddPCR in detecting bacterial pathogens in the blood of COVID-19 critically ill patients. METHODS: This prospective diagnostic study included RT-PCR-confirmed COVID-19 patients admitted to our hospital from December 2022 to February 2023. A multiplex ddPCR assay was used to detect common bacterial pathogens and AMR genes in blood samples of the patients, along with antimicrobial susceptibility testing (AST). The diagnostic performance of the ddPCR assay was evaluated by comparing the results with those obtained through blood culture and clinical diagnosis. Additionally, the ability of ddPCR in detecting bacterial resistance was compared with the AST results. RESULTS: Of the 200 blood samples collected from 184 patients, 45 (22.5%) were positive using blood culture, while 113 (56.5%) were positive for bacterial targets using the ddPCR assay. The ddPCR assay outperformed blood culture in pathogen detection rate, mixed infection detection rate, and fungal detection rate. Acinetobacter baumannii and Klebsiella pneumoniae were the most commonly detected pathogens in COVID-19 critically ill patients, followed by Enterococcus and Streptococcus. Compared to blood culture, ddPCR achieved a sensitivity of 75.5%, specificity of 51.0%, PPV of 30.9%, and NPV of 87.8%, respectively. However, there were significant differences in sensitivity among different bacterial species, where Gram-negative bacteria have the highest sensitivity of 90.3%. When evaluated on the ground of clinical diagnosis, the sensitivity, specificity, PPV and NPV of ddPCR were 78.1%, 90.5%, 94.7%, and 65.5%, respectively. In addition, the ddPCR assay detected 23 cases of blaKPC, which shown a better consistent with clinical test results than other detected AMR genes. Compared to blaKPC, there were few other AMR genes detected, indicating that the application of other AMR gene detection in the COVID-19 critically ill patients was limited. CONCLUSION: The multiplex ddPCR assay had a significantly higher pathogen detection positivity than the blood culture, which could be an effective diagnostic tool for BSIs in COVID-19 patients and to improve patient outcomes and reduce the burden of sepsis on the healthcare system, though there is room for optimization of the panels used.- Adjusting the targets to include E. faecalis and E. faecium as well as Candida albicans and Candida glabrata could improve the ddPCR' s effectiveness. However, further research is needed to explore the potential of ddPCR in predicting bacterial resistance through AMR gene detection.

CDKN2A somatic copy number amplification in normal tissues surrounding gastric carcinoma reduces cancer metastasis risk in droplet digital PCR analysis.

BACKGROUND: The CDKN2A gene is frequently affected by somatic copy number variations (SCNVs, including deletions and amplifications [SCNdel and SCNamp]) in the cancer genome. Using surgical gastric margin tissue samples (SMs) as the diploid reference in SCNV analysis via CDKN2A/P16-specific real-time PCR (P16-Light), we previously reported that the CDKN2A SCNdel was associated with a high risk of metastasis of gastric carcinoma (GC). However, the status of CDKN2A SCNVs in SMs and their clinical significance have not been reported. METHODS: Peripheral white blood cell (WBC) and frozen GC and SM tissue samples were collected from patients (n = 80). Droplet digital PCR (ddPCR) was used to determine the copy number (CN) of the CDKN2A gene in tissue samples using paired WBCs as the diploid reference. RESULTS: A novel P16-ddPCR system was initially established with a minimal proportion (or limit, 10%) of the detection of CDKN2A CN alterations. While CDKN2A SCNamp events were detected in both SMs and GCs, fewer CDKN2A SCNdel events were detected in SMs than in GCs (15.0% vs. 41.3%, P = 4.77E-04). Notably, significantly more SCNamp and fewer SCNdel of the CDKN2A gene were detected in SMs from GC patients without metastasis than in those from patients with lymph node metastasis by P16-ddPCR (P = 0.023). The status of CDKN2A SCNVs in SM samples was significantly associated with overall survival (P = 0.032). No cancer deaths were observed among the 11 patients with CDKN2A SCNamp. CONCLUSION: CDKN2A SCNVs in SMs identified by P16-ddPCR are prevalent and significantly associated with GC metastasis and overall survival.

Exosomal mRNA in plasma serves as a predictive marker for microvascular invasion in hepatocellular carcinoma.

BACKGROUND AND AIM: There is a pressing need for non-invasive preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC). This study investigates the potential of exosome-derived mRNA in plasma as a biomarker for diagnosing MVI. METHODS: Patients with suspected HCC undergoing hepatectomy were prospectively recruited for preoperative peripheral blood collection. Exosomal RNA profiling was conducted using RNA sequencing in the discovery cohort, followed by differential expression analysis to identify candidate targets. We employed multiplexed droplet digital PCR technology to efficiently validate them in a larger sample size cohort. RESULTS: A total of 131 HCC patients were ultimately enrolled, with 37 in the discovery cohort and 94 in the validation cohort. In the validation cohort, the expression levels of RSAD2, PRPSAP1, and HOXA2 were slightly elevated while CHMP4A showed a slight decrease in patients with MVI compared with those without MVI. These trends were consistent with the findings in the discovery cohort, although they did not reach statistical significance (P > 0.05). Notably, the expression level of exosomal PRPSAP1 in plasma was significantly higher in patients with more than 5 MVI than in those without MVI (0.147 vs 0.070, P = 0.035). CONCLUSION: This study unveils the potential of exosome-derived PRPSAP1 in plasma as a promising indicator for predicting MVI status preoperatively.

High-efficiency EGFR genotyping using cell-free DNA in bronchial washing fluid.

BACKGROUND: EGFR mutation testing is required for treatment of lung adenocarcinoma using epidermal growth factor receptor-tyrosine kinase inhibitor. However, the amounts of tumor tissue or tumor cells obtained by bronchoscopy are often insufficient. Bronchial washing fluid, obtained by lavage with saline after tumor biopsy or brushing, and the supernatant of bronchial washing fluid are thought to contain cell-free DNA that would be potentially applicable for EGFR testing. METHODS: From among patients with suspected adenocarcinoma or non-small cell lung carcinoma diagnosed from biopsy or surgical specimens at the University of Tsukuba Hospital between 2015 and 2019, cell-free DNAs from 80 specimens of supernatant of bronchial washing fluid (50 with EGFR mutation and 30 with wild type EGFR) and 8 blood serum samples were examined for EGFR mutation using droplet digital PCR. RESULTS: Among the 50 patients harboring EGFR mutation, the rate of positivity for cell-free DNA extracted from supernatant of bronchial washing fluid was 80% (40/50). In nine of the EGFR mutation-positive cases, tumor cells were not detected by either biopsy or cytology, but the mutation was detected in four cases (4/9, 44%). Comparison of the cell-free DNA mutation detection rate between supernatant of bronchial washing fluid and blood serum in six cases showed that mutations were detected from the former in all cases (6/6, 100%), but from the latter in only one case (1/6, 17%). CONCLUSIONS: Using supernatant of bronchial washing fluid samples, the detection rate of EGFR mutation was high, and EGFR mutations were detectable even when no tumor cells had been detectable by biopsy or cytology. Supernatant of bronchial washing fluid might be an effective sample source for EGFR mutation testing.

To evaluate the performance of simultaneous amplification and testing assay for group B Streptococcus detection: comparison with real-time PCR and ddPCR assays.

BACKGROUND: To evaluate the performance of simultaneous amplification and testing (SAT) assay for the detection of group B Streptococcus (GBS) in maternal vaginal and perianal swabs compared with real-time polymerase chain reaction (RT-PCR). METHODS: We obtained vaginal and perianal swabs from 1474 pregnant women at the Obstetrics and Gynecology Hospital of Fudan University (Shanghai, China) between April 2023 and June 2023. Vaginal and perianal swabs were collected at 35-37 weeks of gestation. Swabs were tested for GBS simultaneously by using the SAT assay and RT-PCR, and a comparative analysis (kappa coefficient) was performed. Furthermore, we conducted additional droplet digital PCR (ddPCR) tests to confirm the results when there were controversial results between SAT and RT-PCR. In addition, we compared the limit of detection, technical specificity, repeatability and reproducibility of SAT-GBS with those of routine RT-PCR assays. RESULTS: In our study, the detection rate of clinical GBS according to the SAT assay was 11.5% (169/1471). The SAT assay showed a sensitivity of 91.8%, a specificity of 99.9%, a diagnostic accuracy of 98.9%, a positive predictive value (PPV) of 99.4% and a negative predictive value (NPV) of 98.8%. The kappa value between RT-PCR and SAT was 0.917. CONCLUSIONS: This SAT assay for the detection of group B Streptococcus is not only easy to perform but can also detect GBS sensitively and specifically and may be used in the regular molecular diagnosis of GBS infection among pregnancies.

Detecting and quantifying Veillonella by real-time quantitative PCR and droplet digital PCR.

Veillonella spp. are Gram-negative opportunistic pathogens present in the respiratory, digestive, and reproductive tracts of mammals. An abnormal increase in Veillonella relative abundance in the body is closely associated with periodontitis, inflammatory bowel disease, urinary tract infections, and many other diseases. We designed a pair of primers and a probe based on the 16S rRNA gene sequences of Veillonella and conducted real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) to quantify the abundance of Veillonella in fecal samples. These two methods were tested for specificity and sensitivity using simulated clinical samples. The sensitivity of qPCR was 100 copies/µL, allowing for the accurate detection of a wide range of Veillonella concentrations from 103 to 108 CFU/mL. The sensitivity of ddPCR was 11.3 copies/µL, only allowing for the accurate detection of Veillonella concentrations from 101 to 104 CFU/mL because of the limited number of droplets generated by ddPCR. ddPCR is therefore more suitable for the detection of low-abundance Veillonella samples. To characterize the validity of the assay system, clinical samples from children with inflammatory bowel disease were collected and analyzed, and the results were verified using isolation methods. We conclude that molecular assays targeting the 16S rRNA gene provides an important tool for the rapid diagnosis of chronic and infectious diseases caused by Veillonella and also supports the isolation and identification of Veillonella for research purposes. KEY POINTS: • With suitable primer sets, the qPCR has a wider detection range than ddPCR. • ddPCR is suitable for the detection of low-abundance samples. • Methods successfully guided the isolation of Veillonella in clinical sample.

Examining the efficiency of porcine gastric mucin-coated magnetic beads in extraction of noroviruses from frozen berries.

Human norovirus is the leading cause of foodborne gastroenteritis worldwide. Due to the low infectious dose of noroviruses, sensitive methodologies are required to detect and characterize small numbers of viral particles that are found in contaminated foods. The ISO 15216 method, which is internationally recognized for detection of foodborne viruses from high-risk food commodities, is based on viral precipitation, followed by RNA extraction and identification of the viral genome by RT-PCR. Although the ISO 15216 method is efficient, it is time consuming and tedious, does not report on the viral infectivity, and is sensitive to the presence of RT-PCR inhibitors. Norovirus capture by the porcine gastric mucin conjugated magnetic beads (PGM-MB) was developed as an alternative virus recovery method. It relies on the integrity of the viral capsid being able to bind to PGM. PGM contains a variety of histo-blood group antigens (HBGAs) that act as norovirus receptors. Therefore, the PGM-MB method allows for extraction of noroviruses, with potentially intact viral capsids, from complex food matrices. The viral genome can then be released through heat-shock of the captured virus. For this reason, we performed a parallel comparison between the ISO 15216 method and the PGM-MB method in isolation and quantification of noroviruses from frozen raspberries. We have demonstrated that the efficiency of the PGM-MB method in extraction of murine norovirus (MNV) and human norovirus GII.4 from raspberries is equal or better than the ISO 15216 method, while the PGM-MB has fewer steps and shorter turnaround time. Moreover, the PGM-MB method is more efficient in removing the inhibitors prior to RT-PCR analysis.

Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05-0.5% within 2 h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.

Expansion of highly interferon-responsive T cells in early-onset Alzheimer's disease.

INTRODUCTION: Altered immune signatures are emerging as a central theme in neurodegenerative disease, yet little is known about immune responses in early-onset Alzheimer's disease (EOAD). METHODS: We examined single-cell RNA-sequencing (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) and droplet digital polymerase chain reaction (ddPCR) data from CD4 T cells from participants with EOAD and clinically normal controls. RESULTS: We analyzed PBMCs from 16 individuals by scRNA-seq and discovered increased interferon signaling-associated gene (ISAG) expression and striking expansion of antiviral-like ISAGhi T cells in EOAD. Isolating CD4 T cells from 19 individuals, including four cases analyzed by scRNA-seq, we confirmed increased expression of ISAGhi marker genes. Publicly available cerebrospinal fluid leukocyte scRNA-seq data from late-onset mild cognitive impairment and AD also revealed increased expression of interferon-response genes. DISCUSSION: Antiviral-like ISAGhi T cells are expanded in EOAD. Additional research into these cells and the role of heightened peripheral IFN signaling in neurodegeneration is warranted. HIGHLIGHTS: Interferon-responsive T cells expanded in early-onset Alzheimer's disease (AD). Increased interferon-associated gene expression present in early- and late-onset AD. Peripheral immune changes in T and NK cells driven by females with early-onset AD.

Plasma miR-203a-3p as a Novel Predictor of Dementia in Patients with Parkinson's Disease.

The early detection of cognitive decline in Parkinson's disease is important for providing drug therapy and non-pharmacological management. The circulating microRNAs present in plasma are promising biomarkers of PD with dementia (PDD) due to their critical roles in synaptic plasticity and the regulation of neurodegeneration-associated proteins. In this study, we aimed to identify plasma microRNAs that may differentiate PD with or without cognitive impairment. Global microRNA expression was obtained from a discovery set of 123 participants who were divided into four groups, namely normal controls (HC), PD with no dementia (PDND), PD with mild cognitive impairment (PD-MCI), and PDD, using next-generation sequencing. The BOLD selector was used for microRNA candidate selection. Six miRNAs, namely miR-203a-3p, miR-626, miR-662, miR-3182, miR-4274, and miR-4295, were clustered as potential candidates for use in identifying PDND from PD-MCI. Another independent cohort of 120 participants was further recruited in a validation step in order to detect candidate microRNAs via droplet digital PCR (ddPCR), which was used for its high sensitivity in detecting low miRNA concentrations. Our results show that the ratio of miR-203a-3p/miR-16-5p, in which miR-16-5p was used as a reference control miRNA, was significantly increased in PDD compared to that seen in PD-MCI and PDND individually, and was negatively correlated with the MoCA scores (r = -0.237, p = 0.024) in patients with PD. However, there was no significant difference in the ratio of miR-203a-3p/miR-16-5p between HC and PDND, PD-MCI, or PDD individually. The ROC curve of the logistic regression model, factoring in the variables of age, the ratio of miR-203a-3p/miR-16-5p, and the UPDRS III score, demonstrated an AUC of 0.883. Our findings suggest that the ratio of miR-203a-3p/miR-16-5p, used with age and motor score, could be a predictor of dementia among PD patients.