Corrigendum: Optimization of tomato (Solanum lycopersicum L.) juice fermentation process and analysis of its metabolites during fermentation.

[This corrects the article DOI: 10.3389/fnut.2024.1344117.].

Potential Role of Lauric Acid in Milk Fat Synthesis in Chinese Holstein Cows Based on Integrated Analysis of Ruminal Microbiome and Metabolome.

The composition and metabolic profile of the ruminal microbiome have an impact on milk composition. To unravel the ruminal microbiome and metabolome affecting milk fat synthesis in dairy cows, 16S rRNA and internal transcribed spacer (ITS) gene sequencing, as well as ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods were used to investigate the significant differences in ruminal bacterial and fungal communities as well as metabolome among Chinese Holstein cows with contrasting milk fat contents under the same diet (H-MF 5.82 ± 0.41% vs. L-MF 3.60 ± 0.12%). Another objective was to culture bovine mammary epithelial cells (BMECs) to assess the effect of metabolites on lipid metabolism. Results showed that the acetate-to-propionate ratio and xylanase activity in ruminal fluid were both higher in H-MF. Microbiome sequencing identified 10 types of bacteria and four types of fungi differently abundant at the genus level. Metabolomics analysis indicated 11 different ruminal metabolites between the two groups, the majority of which were lipids and organic acids. Among these, lauric acid (LA) was enriched in fatty acid biosynthesis with its concentration in milk fat of H-MF cows being greater (217 vs. 156 mg per 100 g milk), thus, it was selected for an in vitro study with BMECs. Exogenous LA led to a marked increase in intracellular triglyceride (TG) content and lipid droplet formation, and it upregulated the mRNA abundance of fatty acid uptake and activation (CD36 and ACSL1), TG synthesis (DGAT1, DGAT2 and GPAM), and transcriptional regulation (SREBP1) genes. Taken together, the greater relative abundance of xylan-fermenting bacteria and fungi, and lower abundance of bacteria suppressing short-chain fatty acid-producing bacteria or participating in fatty acid hydrogenation altered lipids and organic acids in the rumen of dairy cows. In BMECs, LA altered the expression of genes involved in lipid metabolism in mammary cells, ultimately promoting milk fat synthesis. Thus, it appears that this fatty acid plays a key role in milk fat synthesis.

A wild melon reference genome provides novel insights into the domestication of a key gene responsible for melon fruit acidity.

KEY MESSAGE: A wild melon reference genome elucidates the genomic basis of fruit acidity domestication. Structural variants (SVs) have been reported to impose major effects on agronomic traits, representing a significant contributor to crop domestication. However, the landscape of SVs between wild and cultivated melons is elusive and how SVs have contributed to melon domestication remains largely unexplored. Here, we report a 379-Mb chromosome-scale genome of a wild progenitor melon accession "P84", with a contig N50 of 14.9 Mb. Genome comparison identifies 10,589 SVs between P84 and four cultivated melons with 6937 not characterized in previously analysis of 25 melon genome sequences. Furthermore, the population-scale genotyping of these SVs was determined in 1175 accessions, and 18 GWAS signals including fruit acidity, fruit length, fruit weight, fruit color and sex determination were detected. Based on these genotyped SVs, we identified 3317 highly diverged SVs between wild and cultivated melons, which could be the potential SVs associated with domestication-related traits. Furthermore, we identify novel SVs affecting fruit acidity and proposed the diverged evolutionary trajectories of CmPH, a key regulator of melon fruit acidity, during domestication and selection of different populations. These results will offer valuable resources for genomic studies and genetic improvement in melon.

Ethylene-responsive VviERF003 modulates glycosylated monoterpenoid synthesis by upregulating VviGT14 in grapes.

Terpenoids are important contributors to the aroma of grapes and wines. Grapes contain terpenoids in both volatile free form and non-volatile glycosidic form, with the latter being more abundant. Glycosylated terpenoids are deemed as latent aromatic potentials for their essential role in adding to the flowery and fruity bouquet of wines. However, the transcriptional regulatory mechanism underlying glycosylated terpenoid biosynthesis remains poorly understood. Our prior study identified an AP2/ERF transcription factor, VviERF003, through DNA pull-down screening using the promoter of terpenoid glycosyltransferase VviGT14 gene. This study demonstrated that both genes were co-expressed and synchronized with the accumulation of glycosylated monoterpenoids during grape maturation. VviERF003 can bind to the VviGT14 promoter and promote its activity according to yeast one-hybrid and dual-luciferase assays. VviERF003 upregulated VviGT14 expression in vivo, leading to increased production of glycosylated monoterpenoids based on the evidence from overexpression or RNA interference in leaves, berry skins, and calli of grapes, as well as tomato fruits. Additionally, VviERF003 and VviGT14 expressions and glycosylated monoterpenoid levels were induced by ethylene in grapes. The findings suggest that VviERF003 is ethylene-responsive and stimulates glycosylated monoterpenoid biosynthesis through upregulating VviGT14 expression.

Effects of EGFR-TKI on epidermal melanin unit integrity: Therapeutic implications for hypopigmented skin disorders.

Epidermal melanin unit integrity is crucial for skin homeostasis and pigmentation. Epidermal growth factor (EGF) receptor (EGFR) is a pivotal player in cell growth, wound healing, and maintaining skin homeostasis. However, its influence on skin pigmentation is relatively unexplored. This study investigates the impact and underlying mechanisms of EGFR inhibitors on skin pigmentation. We evaluated EGF and EGFR expression in various skin cells using quantitative real-time PCR, Western blot, and immunofluorescence. EGF and EGFR were predominantly expressed in epidermal keratinocytes, and treatment with the EGFR tyrosine kinase inhibitors (EGFR-TKIs) gefitinib and PD153035 significantly increased stem cell factor (SCF) and endothelin-1 (ET-1) expression in cultured keratinocytes. Enhanced melanocyte migration and proliferation were observed in co-culture, as evidenced by time-lapse live imaging and single-cell tracking assays. Furthermore, topical application of gefitinib to guinea pig dorsal skin induced increased pigmentation and demonstrated efficacy in mitigating rhododendrol-induced leukoderma. Suppression of EGF signaling indirectly enhanced skin pigmentation by upregulating SCF and ET-1 in epidermal keratinocytes. This novel mechanism highlights the pivotal role of EGF signaling in regulating skin pigmentation, and topical EGFR-TKI therapy at an appropriate dose may be a promising approach for depigmentation disorder management.

Correction: Expanded graphite incorporated with Li4Ti5O12 nanoparticles as a high-rate lithium-ion battery anode.

[This corrects the article DOI: 10.1039/D4RA00832D.].

Modulation Steering Motion by Quantitative Electrical Stimulation in Pigeon Robots.

The pigeon robot has attracted significant attention in the field of animal robotics thanks to its outstanding mobility and adaptive capability in complex environments. However, research on pigeon robots is currently facing bottlenecks, and achieving fine control over the motion behavior of pigeon robots through brain-machine interfaces remains challenging. Here, we systematically quantify the relationship between electrical stimulation and stimulus-induced motion behaviors, and provide an analytical method to demonstrate the effectiveness of pigeon robots based on electrical stimulation. In this study, we investigated the influence of gradient voltage intensity (1.2-3.0 V) on the indoor steering motion control of pigeon robots. Additionally, we discussed the response time of electrical stimulation and the effective period of the brain-machine interface. The results indicate that pigeon robots typically exhibit noticeable behavioral responses at a 2.0 V voltage stimulus. Increasing the stimulation intensity significantly controls the steering angle and turning radius (p < 0.05), enabling precise control of pigeon robot steering motion through stimulation intensity regulation. When the threshold voltage is reached, the average response time of a pigeon robot to the electrical stimulation is 220 ms. This study quantifies the role of each stimulation parameter in controlling pigeon robot steering behavior, providing valuable reference information for the precise steering control of pigeon robots. Based on these findings, we offer a solution for achieving precise control of pigeon robot steering motion and contribute to solving the problem of encoding complex trajectory motion in pigeon robots.

A nomogram for predicting pathologic node negativity after neoadjuvant chemotherapy in breast cancer patients: a nationwide, multicenter retrospective cohort study (CSBrS-012).

Purpose: This study aimed to investigate the factors associated with pathologic node-negativity (ypN0) in patients who received neoadjuvant chemotherapy (NAC) to develop and validate an accurate prediction nomogram. Methods: The CSBrS-012 study (2010-2020) included female patients with primary breast cancer treated with NAC followed by breast and axillary surgery in 20 hospitals across China. In the present study, 7,711 eligible patients were included, comprising 6,428 patients in the primary cohort from 15 hospitals and 1,283 patients in the external validation cohort from five hospitals. The hospitals were randomly assigned. The primary cohort was randomized at a 3:1 ratio and divided into a training set and an internal validation set. Univariate and multivariate logistic regression analyses were performed on the training set, after which a nomogram was constructed and validated both internally and externally. Results: In total, 3,560 patients (46.2%) achieved ypN0, and 1,558 patients (20.3%) achieved pathologic complete response in the breast (bpCR). A nomogram was constructed based on the clinical nodal stage before NAC (cN), ER, PR, HER2, Ki67, NAC treatment cycle, and bpCR, which were independently associated with ypN0. The area under the receiver operating characteristic curve (AUC) for the training set was 0.80. The internal and external validation demonstrated good discrimination, with AUCs of 0.79 and 0.76, respectively. Conclusion: We present a real-world study based on nationwide large-sample data that can be used to effectively screen for ypN0 to provide better advice for the management of residual axillary disease in breast cancer patients undergoing NAC.

Convolutional neural network with parallel convolution scale attention module and ResCBAM for breast histology image classification.

Breast cancer is the most common cause of female morbidity and death worldwide. Compared with other cancers, early detection of breast cancer is more helpful to improve the prognosis of patients. In order to achieve early diagnosis and treatment, clinical treatment requires rapid and accurate diagnosis. Therefore, the development of an automatic detection system for breast cancer suitable for patient imaging is of great significance for assisting clinical treatment. Accurate classification of pathological images plays a key role in computer-aided medical diagnosis and prognosis. However, in the automatic recognition and classification methods of breast cancer pathological images, the scale information, the loss of image information caused by insufficient feature fusion, and the enormous structure of the model may lead to inaccurate or inefficient classification. To minimize the impact, we proposed a lightweight PCSAM-ResCBAM model based on two-stage convolutional neural network. The model included a Parallel Convolution Scale Attention Module network (PCSAM-Net) and a Residual Convolutional Block Attention Module network (ResCBAM-Net). The first-level convolutional network was built through a 4-layer PCSAM module to achieve prediction and classification of patches extracted from images. To optimize the network's ability to represent global features of images, we proposed a tiled feature fusion method to fuse patch features from the same image, and proposed a residual convolutional attention module. Based on the above, the second-level convolutional network was constructed to achieve predictive classification of images. We evaluated the performance of our proposed model on the ICIAR2018 dataset and the BreakHis dataset, respectively. Furthermore, through model ablation studies, we found that scale attention and dilated convolution play an important role in improving model performance. Our proposed model outperforms the existing state-of-the-art models on 200 × and 400 × magnification datasets with a maximum accuracy of 98.74 %.

Phase I Trial of GD2.CART Cells Augmented With Constitutive Interleukin-7 Receptor for Treatment of High-Grade Pediatric CNS Tumors.

PURPOSE: T cells modified with chimeric antigen receptors (CARTs) have demonstrated efficacy for hematologic malignancies; however, benefit for patients with CNS tumors has been limited. To enhance T cell activity against GD2+ CNS malignancies, we modified GD2-directed CART cells (GD2.CARTs) with a constitutively active interleukin (IL)-7 receptor (C7R-GD2.CARTs). METHODS: Patients age 1-21 years with H3K27-altered diffuse midline glioma (DMG) or other recurrent GD2-expressing CNS tumors were eligible for this phase I trial (ClinicalTrials.gov identifier: NCT04099797). All subjects received standard-of-care adjuvant radiation therapy or chemotherapy before study enrollment. The first treatment cohort received GD2.CARTs alone (1 × 107 cells/m2), and subsequent cohorts received C7R-GD2.CARTs at two dose levels (1 × 107 cells/m2; 3 × 107 cells/m2). Standard lymphodepletion with cyclophosphamide and fludarabine was included at all dose levels. RESULTS: Eleven patients (age 4-18 years) received therapy without dose-limiting toxicity. The GD2.CART cohort did not experience toxicity, but had disease progression after brief improvement of residual neurologic deficits (≤3 weeks). The C7R-GD2.CART cohort developed grade 1 tumor inflammation-associated neurotoxicity in seven of eight (88%) cases, controllable with anakinra. Cytokine release syndrome was observed in six of eight (75%, grade 1 in all but one patient) and associated with increased circulating IL-6 and IP-10 (P < .05). Patients receiving C7R-GD2.CARTs experienced temporary improvement from baseline neurologic deficits (range, 2 to >12 months), and seven of eight (88%) remained eligible for additional treatment cycles (range 2-4 cycles). Partial responses by iRANO criteria were observed in two of seven (29%) patients with DMG treated by C7R-GD2.CARTs. CONCLUSION: Intravenous GD2.CARTs with and without C7R were well tolerated. Patients treated with C7R-GD2.CARTs exhibited transient improvement of neurologic deficits and increased circulating cytokines/chemokines. Treatment with C7R-GD2.CARTs represents a novel approach warranting further investigation for children with these incurable CNS cancers.

CmBr confers fruit bitterness under CPPU treatment in melon.

Many biotic or abiotic factors such as CPPU (N-(2-chloro-pyridin-4-yl)-N'-phenylurea), a growth regulator of numerous crops, can induce bitterness in cucurbits. In melon, cucurbitacin B is the major compound leading to bitterness. However, the molecular mechanism underlying CuB biosynthesis in response to different conditions remains unclear. Here, we identified a set of genes involved in CPPU-induced CuB biosynthesis in melon fruit and proposed CmBr gene as the major regulator. Using CRISPR/Cas9 gene editing, we confirmed CmBr's role in regulating CuB biosynthesis under CPPU treatment. We further discovered a CPPU-induced MYB-related transcription factor, CmRSM1, which specifically binds to the Myb motif within the CmBr promoter and activates its expression. Moreover, we developed an introgression line by introducing the mutated Cmbr gene into an elite variety and eliminated CPPU-induced bitterness, demonstrating its potential application in breeding. This study offers a valuable tool for breeding high-quality non-bitter melon varieties and provides new insights into the regulation of secondary metabolites under environmental stresses.

Case report: Dual dabrafenib and trametinib therapy for treating BRAF V600E mutated lung adenocarcinoma with BRCA2 germline mutation post multiline progression.

The v-raf murine sarcoma viral oncogenic homolog B1 (BRAF) V600E is a rare mutation that functions as an oncogenic driver in patients with non-small cell lung cancer (NSCLC) leading to the overactivation of the RAS-RAF-MEK-ERK (MAPK) pathway and the subsequent uncontrolled cell proliferation. Understanding the mechanism behind BRAF mutation, its inhibition, and relationship to the upstream and downstream effector is essential for advancing treatment strategies for NSCLC patients with the BRAF V600E mutation. Next-generation sequencing studies have identified the presence of breast cancer susceptibility gene 1/2 (BRCA1/2) mutations in NSCLC patients, which are pathogenic variants associated with breast, ovarian, and prostate cancers. Although poly ADP-ribose polymerase (PARP) inhibitors are currently an approved treatment option for malignant tumors linked to BRCA1/2 pathogenic variants, the therapeutic potential of PARP inhibitors in NSCLC remains unclear. The development of genetic testing provides a platform for investigating the pathophysiological mechanisms of genetic mutations above. Here, we report a novel case of a middle-aged non-smoking female diagnosed with BRAF V600E and BRCA2 germline mutated lung adenocarcinoma, who had previously undergone a diverse array of cancer-targeted therapies, including PARP inhibitor, before the identification of the BRAF V600E mutation. Following this, a combination of dabrafenib and trametinib was administered and induced a rapid and positive response within two months. Our case not only highlights the importance of dynamic and repetitive genetic testing in managing patients, but contributes to the growing body of clinical evidence supporting the efficacy of BRAF/MEK co-inhibition in patients harboring a BRAF V600E mutation and provokes thinking for further research into the impact of PARP inhibitors in BRCA1/2-mutated NSCLC.

Prognostic value of pathological nodal burden after neoadjuvant chemotherapy in initially cN0-1 breast cancer patients: a dual-center, 10-year survival analysis.

Background: There is an interest in performing de-escalating axillary surgery after neoadjuvant chemotherapy (NAC). However, the significance of residual axillary node disease after NAC has not been well studied. Objectives: To investigate the pathological residual axillary lymph node tumor burden (ypN) of patients with initial clinical nodal stage cN0-1 breast cancer after NAC and determine its prognostic value. Design: Initial cN0-1 breast cancer patients who received NAC followed by axillary surgery at the First Hospital of Jilin University and the First Affiliated Hospital of Xi'an Jiaotong University between January 2011 and December 2019 were included. Methods: Survival outcomes were compared according to different clinical and pathological stage and nodal response to NAC. The main outcomes were disease-free survival (DFS) and overall survival (OS). Factors associated with survival were defined by Cox regression analysis. Results: A total of 911 patients were included, among whom 260 had cN0 and 651 had cN1 tumors. After NAC, 410 patients were ypN0, and another 501 were ypN+. The median follow-up time was 63 months. There was no significant difference in DFS or OS between the cN0 and cN1 groups in hormone receptor positive (HR+)/human epidermal growth factor receptor 2 positive (HER2+) and HR-/HER2- subtypes; instead, ypN status was significantly related to DFS and OS. In HR+/HER2- subtype, both cN and ypN stages did not show significant survival differences, but the ypN number and the nodal response to NAC showed significant prognostic value (p < 0.05). Among HR-/HER2+ patients, all cN status, ypN status, ypN number, and nodal response were significantly associated with survival (p < 0.05). Furthermore, tumor biology, axillary surgery, ypN status, pathological tumor size, and radiotherapy were independent prognostic factors for DFS and OS. Conclusion: The ypN status after NAC provide more prognostic information than the initial cN stage in cN0-1 patients, and the surgical axillary staging after NAC may have high clinical value.

The combination of breast cancer PDO and mini-PDX platform for drug screening and individualized treatment.

The majority of advanced breast cancers exhibit strong aggressiveness, heterogeneity, and drug resistance, and currently, the lack of effective treatment strategies is one of the main challenges that cancer research must face. Therefore, developing a feasible preclinical model to explore tailored treatments for refractory breast cancer is urgently needed. We established organoid biobanks from 17 patients with breast cancer and characterized them by immunohistochemistry (IHC) and next generation sequencing (NGS). In addition, we in the first combination of patient-derived organoids (PDOs) with mini-patient-derived xenografts (Mini-PDXs) for the rapid and precise screening of drug sensitivity. We confirmed that breast cancer organoids are a high-fidelity three-dimension (3D) model in vitro that recapitulates the original tumour's histological and genetic features. In addition, for a heavily pretreated patient with advanced drug-resistant breast cancer, we combined PDO and Mini-PDX models to identify potentially effective combinations of therapeutic agents for this patient who were alpelisib + fulvestrant. In the drug sensitivity experiment of organoids, we observed changes in the PI3K/AKT/mTOR signalling axis and oestrogen receptor (ER) protein expression levels, which further verified the reliability of the screening results. Our study demonstrates that the PDO combined with mini-PDX model offers a rapid and precise drug screening platform that holds promise for personalized medicine, improving patient outcomes and addressing the urgent need for effective therapies in advanced breast cancer.

Identification and Characterization of long non-coding RNAs in Mammary Gland Tissues of Chinese Holstein Cows.

LncRNAs (Long non-coding RNA) is an RNA molecule with a length more than 200bp. LncRNAs can directly act on mRNA, thus affecting the expression of downstream target genes and proteins, and widely participate in many important physiological and pathological regulation processes of the body. In this study, RNA-Seq was performed to detect lncRNAs from mammary gland tissues of 3 Chinese Holstein cows, including 3 cows at 7 days before calving and the same 3 cows at 30 days postpartum (early lactation stage). A total of 1,905 novel lncRNAs were detected, 57.3% of the predicted lncRNAs are ≥ 500bp and 612 lncRNAs are intronic lncRNAs. The exon number of lncRNAs ranged from 2 to 10. A total of 96 lncRNAs were significantly differentially expressed between two stages, which 47 were upregulated and 49 were downregulated. Pathway analysis found that target genes were mainly concentrated on the ECM-receptor interaction, Jak-STAT signaling pathway, PI3K-Akt signaling pathway and TGF-beta signaling pathway. This study revealed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows at non-lactation and early lactation period, and providing a basis for studying the functions of lncRNAs in Holstein cows during different lactation periods.

Severe fever with thrombocytopenia syndrome with central nervous system symptom onset: a case report and literature review.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a natural focal disease transmitted mainly by tick bites, and the causative agent is SFTS virus (SFTSV). SFTS can rapidly progress to severe disease, with multiple-organ failure (MOF) manifestations such as shock, respiratory failure, disseminated intravascular coagulation (DIC) and death, but cases of SFTS patients with central nervous system (CNS) symptoms onset and marked persistent involuntary shaking of the perioral area and limbs have rarely been reported. CASE PRESENTATION: A 69-year-old woman with fever and persistent involuntary shaking of the perioral area and limbs was diagnosed with SFTS with CNS symptom onset after metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) and peripheral blood identified SFTSV. The patient developed a cytokine storm and MOF during the course of the disease, and after aggressive antiviral, glucocorticoid, and gamma globulin treatments, her clinical symptoms improved, her laboratory indices returned to normal, and she had a good prognosis. CONCLUSION: This case gives us great insight that when patients with CNS symptoms similar to those of viral encephalitis combined with thrombocytopenia and leukopenia are encountered in the clinic, it is necessary to consider the possibility of SFTS involving the CNS. Testing for SFTSV nucleic acid in CSF and blood (mNGS or polymerase chain reaction (PCR)) should be carried out, especially in critically ill patients, and treatment should be given accordingly.

Aptamer-RIBOTAC Strategy Enabling Tumor-Specific Targeted Degradation of MicroRNA for Precise Cancer Therapy.

MicroRNA (miRNA) molecules play crucial roles in a variety of diseases, making miRNA targeting a burgeoning field in medicinal chemistry. Ribonuclease targeting chimeras (RIBOTACs) present a compelling approach for RNA degradation. However, small molecule-based RIBOTAC requires an expensive and time-consuming screening process, and is difficult to directly target miRNA due to its short length lacking secondary structure. Antisense oligonucleotide (ASO)-based RIBOTAC is easy to design but with poor cell permeability. While both of them lack the specificity for tumor targeting. In this study, the first Aptamer-RIBOTAC (ARIBOTAC) chimera is designed based on ASO to achieve precise degradation of miRNA in a tumor cell-specific manner for precise cancer therapy. This chimera exhibits a remarkable ability to specifically identify and enter cancer cells, trigger localized activation of endogenous RNase L, and selectively cleave miRNAs that are complementary to ASO. The efficacy and universality of the ARIBOTAC strategy both in vitro and in vivo by degrading oncogenic miR-210-3p and miR-155-5p are validated. These findings underscore the potential of the ARIBOTAC strategy as a promising avenue for cancer therapy by precisely targeting cancer-associated miRNAs.

Novel target and PCR assay for identification of hypervirulent ST1 (BI/NAP1/027) Clostridioides difficile and detection of toxigenic C. Difficile.

BACKGROUND AND AIMS: The incidence of Clostridioides difficile infection and the prevalence of hypervirulent ST1 (BI/NAP1/027)strain are increasing, especially in developing countries. We aimed to develop a new PCR assay for the identification of hypervirulent ST1 strains and toxigenic C. difficile in stool samples. MATERIALS AND METHODS: We established a quadruplex TaqMan real-time PCR (pilW_4-plex PCR) assay targeting the pilW, a ST1-specific type Ⅳ minor pilin gene, and three C. difficile genes including cdtB, tcdB, and hsp. The sensitivity and specificity of the assay was tested using 403C. difficile isolates and 180 unformed stool sample. The results were compared with anaerobic culture-based conventional PCR method and MLST. RESULTS: The pilW_4-plex PCR identified toxigenic C. difficile in 333 (82.6%, 333/403) isolates with 100% sensitivity and specificity, and in 78 (43.3%, 78/180) stool samples with the sensitivity and specificity of 94.7% and 93.3%, respectively. Hypervirulent ST1 were detected in 21 strains and nine stool samples by the pilW_4-plex PCR. The pilW_4-plex PCR assay has no cross-reaction with non-toxigenic C. difficile or other bacteria. CONCLUSION: The pilW_4-plex PCR assay is an accurate and rapid method with high sensitivity and specificity for identification of ST1 and detection of toxigenic C. difficile in stool.