Molecular Diagnosis & Therapy最新文献

Introduction: Sepsis is a critical condition requiring timely and accurate pathogen identification. Traditional blood cultures are slow and often yield low sensitivity. Metagenomic next-generation sequencing (mNGS) offers broad and rapid pathogen detection but is hindered by excessive human DNA background in blood samples. This study evaluated a novel Zwitterionic Interface Ultra-Self-assemble Coating (ZISC)-based filtration device designed to deplete host cells and enhance microbial DNA recovery for improved mNGS diagnostics.

Methods: We assessed the novel filter's performance in depleting white blood cells (WBCs) while preserving microbial integrity using spiked blood samples. Comparisons were made with other host depletion techniques, including differential lysis and CpG-methylated DNA removal. Analytical sensitivity was tested using spiked microbial communities at varying genome equivalents (GEs). Clinical validation involved eight blood culture-positive sepsis patient samples, processed with and without filtration, for both genomic DNA (gDNA) and cell-free DNA (cfDNA)-based mNGS. All libraries were sequenced on a NovaSeq600 with at least 10 million reads per sample.

Results: The novel filter achieved > 99% WBC removal across various blood volumes and allowed unimpeded passage of bacteria and viruses. Compared to other depletion methods, the novel filtration was more efficient, less labor-intensive, and preserved microbial reads. mNGS with filtered gDNA detected all expected pathogens in 100% (8/8) of clinical samples, with an average microbial read count of 9351 reads per million (RPM), over tenfold higher than unfiltered samples (925 RPM). In contrast, cfDNA-based mNGS showed inconsistent sensitivity and was not significantly enhanced by filtration (1251-1488 RPM). Finally, the novel filtration did not alter the microbial composition, making it suitable for accurate pathogen profiling.

Conclusion: The workflow with the novel host depletion method significantly enhanced the analytical sensitivity of gDNA-based mNGS by reducing the host DNA background and enriching microbial content. This approach improved diagnostic yield in sepsis and may be a valuable tool for further clinical infectious disease diagnostics.

Circulating tumor DNA has emerged as a minimally invasive and dynamic tool for providing real-time genetic insights into solid tumors, including breast cancer. Circulating tumor DNA is released into the bloodstream through apoptosis, necrosis, or active secretion, and it reflects tumor heterogeneity, which continues to be a major challenge in breast cancer treatment. Advances in high-sensitivity technologies, such as next-generation sequencing and digital polymerase chain reaction, have enabled the detection of key genetic alterations, offering applications in early diagnosis, monitoring minimal residual disease, identifying drug resistance mechanisms, and predicting relapse. Some circulating tumor DNA-based tests have already received regulatory approval for clinical use in patients with breast cancer, and additional studies are underway to expand their applicability. However, low concentrations of circulating tumor DNA and the necessity for standardization across different platforms remain a challenge for the expanded application. In this review, we present an overview of the genetic variants detected in circulating tumor DNA from patients with breast cancer, emphasizing their potential utility in guiding personalized therapeutic strategies and predicting treatment responses across the diverse molecular subtypes of the disease.

The management of spasticity poses a significant challenge for both physicians and patients. This condition, often characterized by increased muscle tone, clonus, and muscle spasms, can be painful, disrupt daily activities, and increases the risk of injuries. First-line treatment for spasticity includes oral medications, such as baclofen, but these drugs can have significant side effects due to inhibitory effects on neural circuits, such as drowsiness and dizziness. Surgical approaches can be applied to patients for whom oral medications fail. However, these treatments can have life-threatening side effects or are irreversible. An emerging technology, chemogenetics, has the potential to provide targeted relief of spasticity, combining a non-destructive surgical approach with the convenience of an oral medication. This Current Opinion describes the current treatment approaches for spasticity, the pathophysiology of this condition, the current state of chemogenetics, and how this technology may be applied to patients with poorly controlled spasticity. Although the use of this approach is at an early developmental stage, we believe that it shows great promise.

Background: Glycogen storage disease type 1b (GSD 1b) is an ultra-rare disease worldwide, whereas in Serbia it has an unexpectedly high prevalence. GSD 1b is the result of variants in the SLC37A4 gene and reduced function of the enzyme glucose 6 phosphate translocase (G6PT). In addition to the classic symptoms of GSD 1a, patients with GSD 1b have neutropenia and impaired neutrophil function.

Methods: The genotype and clinical profile were analyzed in 35 patients, 26 of whom were children. In all patients, pathogenic variants in the SLC37A4 gene were confirmed using Sanger or next-generation sequencing (NGS). Eight different variants were found. The following clinical data were analyzed: age at diagnosis, first symptoms of GSD 1b, severity of intestinal symptoms, lowest neutrophil count, mean hemoglobin value, height, body mass index (BMI), and quality of life. Patients were classified into four groups based on the severity of their intestinal symptoms.

Results: In our study 30 patients received empagliflozin therapy. Our data are comprised of information from a total of 62 treatment years and include self-reported quality-of-life surveys before and during empagliflozin therapy. The average age at which empagliflozin was introduced in pediatric patients was 8.5 years, with the youngest two patients, both female, starting SGLT2 inhibitor therapy at the age of two.

Conclusions: Our findings suggest that empagliflozin therapy significantly improves neutropenia recovery by reducing the frequency of recurrent infections and inflammatory bowel disease (IBD)-like symptoms. This improvement was demonstrated by a marked reduction in skin and mucosal infections, particularly oral ulcers, as well as an increase in hemoglobin levels and overall stature.

Persistent infection with high-risk human papillomavirus (HPV) is a significant factor in cervical cancer (CC) development. Although CC screening programs have reduced the incidence of this neoplasm, the number of deaths remains high, especially in developing countries: CC remains the fourth most common neoplasm in the female population globally. Currently, an HPV test has been replacing cytological analysis because it is a more sensitive screening method. However, the collection of gynecological material is still necessary, which can be a barrier to adherence to testing in the target population. Host cells presenting with a viral infection release fragments of their DNA into circulation, known as cell-free DNA (cfDNA); this allows detection through venous puncture, a routine procedure in clinical laboratories. Thus, the objective of this review was to evaluate the role of cfDNA of HPV (cfHPV-DNA) as an alternative tool for CC screening, diagnosis, prognosis, and treatment response monitoring. Furthermore, the development of sensitive methods, such as droplet digital PCR (ddPCR) and next-generation sequencing (NGS), have proven useful in identifying tumor markers for CC. The specificity of the primers, the size of the target DNA fragments, and variables such as sample type and volume, in addition to the cfDNA extraction kit used, can influence the results of cfHPV-DNA detection. Although the detection of cfHPV-DNA in plasma and serum of patients with CC is feasible, there were conflicting results regarding cfHPV-DNA detection in the blood circulation of patients with premalignant lesions. On the other hand, when CC is already established, the detection and quantification of cfHPV-DNA have shown potential as a biomarker for tumor staging, prognosis definition, and treatment response monitoring.

Background: Molecular diagnostic rates for hereditary hearing loss vary by genetic ancestry, highlighting the importance of population-specific studies. In Pakistan, where consanguineous marriages are prevalent, genetic research has identified many autosomal recessive genes, advancing understanding of rare and novel hearing loss mechanisms. This study aimed to identify pathogenic genetic variants in 31 families from Azad Kashmir, Pakistan, presenting non-syndromic hearing loss.

Methods: We conducted exome sequencing and bioinformatics analysis, and targeted gene sequencing on 31 Pakistani families with hearing loss.

Results: We identified ten pathogenic, three likely pathogenic variants, and one variant of uncertain significance, comprising six nonsense, four missense, three frameshift, and one deep intronic variant, across ten hearing loss-associated genes (MYO15A, GJB2, SLC26A4, TMC1, HGF, TMIE, SLC19A2, KCNE1, ILDR, PCDH15 and MYO6) in 25 families. The overall diagnostic rate, including families with pathogenic and likely pathogenic variants, was 77.4%. GJB2 was the most frequently affected gene, identified in seven families. Thirteen out of 14 identified variants were homozygous. Notably, we identified two novel variants: MYO15A (NM_016239.4, DFNB3) c.870C>G, p.(Tyr290*) and MYO6 (NM_016239.4, DFNB37) c.3465del, p.(Pro1156Leufs*9). Additionally, we identified c.10475dupA, p.(Leu3493Alafs*25) in MYO15A (NM_016239.4, DFNB3) and c.617T>A, p.(Leu206*) in SLC26A4 (NM_000441.2, DFNB4), previously documented in ClinVar but unpublished. We also propose SLC19A2 as a candidate gene presenting as non-syndromic hearing loss, despite its association with thiamine-responsive megaloblastic anemia syndrome.

Conclusion: Our work expands the genotypic and phenotypic spectrum of hearing loss by emphasizing the importance of investigating under-represented groups to identify unique genetic variants and clinical characteristics. Such efforts deepen understanding of genetic diversity in under-represented populations to improve diagnosis and treatment strategies.

The CRISPR/Cas system has been extensively used in the fields of biology, food safety, and environmental monitoring. This is in part because its can to be used in combination with isothermal amplification-mediated signal amplification technology along with its extraordinary trans-cleavage ability, which has initiated a new era of biosensing applications. The popularity of functional nucleic acids has enabled aptamers to convert non-nucleic acid substances into programmable nucleic acid sequences through methods such as direct detection, lock activation, sandwich design, induction of conformations, and split aptamers. Additionally, CRISPR/Cas systems have been extended beyond nucleic acid detection to include ions, small molecules, proteins, cells, bacteria, viruses, and other non-nucleic acid-based target substances. This article provides a brief overview of the mechanisms of action of four Cas proteins, the generation of aptamers, and their combined applications. Moreover, we focus on the research progress of biosensors based on aptamer-based signal conversion combined with the CRISPR/Cas system.

Post-ischemic myocardial remodeling significantly impacts clinical outcomes after acute myocardial infarction (MI), involving structural and functional changes such as ventricular dilation, infarct wall thinning, and fibrosis development. These processes, driven by inflammatory cascades, neurohormonal activation, and extracellular matrix remodeling, result in impaired cardiac output and an increased risk of heart failure. Imaging with fibroblast activation protein inhibitors (FAPI) has emerged as a promising non-invasive tool for assessing myocardial fibrosis via positron emission tomography (PET) or single-photon emission computed tomography (SPECT), targeting activated fibroblasts; the mediators of reparative and fibrotic processes. This innovative approach enables precise visualization and quantification of fibrosis dynamics, surpassing traditional imaging modalities. Preclinical studies using [⁶⁸Ga]Ga-FAPI PET/computed tomography (CT) demonstrated the tracer's specificity for fibroblast activation and its peak uptake in the infarct border zone at day 6 post-MI. These findings, corroborated by histology and autoradiography, highlight its potential for tracking reparative fibrosis. Clinical translation of FAPI imaging was recently achieved with [⁶⁸Ga]Ga-FAPI-46 PET/magnetic resonance imaging (MRI), showing persistent fibroblast activity beyond infarct zones and strong correlations with myocardial injury markers. Complementary research on [^99mTc]Tc-HFAPi SPECT imaging in patients post-MI established its predictive value for left ventricular remodeling, emphasizing its cost-effectiveness and accessibility compared with PET. These advancements underscore FAPI-based imaging's potential to transform risk stratification and therapeutic guidance in post-MI care.

Nectin cell adhesion molecule 4 (Nectin-4) is specifically overexpressed in most cancers of epithelial origin but downregulated in normal tissue, representing an ideal target for positron emission tomography imaging. The development of positron emission tomography imaging probes targeting Nectin-4 has gained significant attention in recent years, especially after the approval in December 2019 by the US Food and Drug Administration of enfortumab vedotin-an antibody drug conjugate targeting Nectin-4-in patients with locally advanced or metastatic bladder cancer. This article aims to comprehensively review original research articles discussing preclinical development or early translational clinical applications of radiolabeled probes targeting Nectin-4. The main radioactive compounds investigated belong to two classes, antibody-based radiopharmaceuticals and peptide-drug conjugates, in particular novel bicyclic peptides. While monoclonal antibody-based probes have demonstrated theranostic potential in preclinical studies, their clinical application has been hindered by their slow pharmacokinetic properties. However, peptide-based positron emission tomography/computed tomography tracers offer several advantages, such as ease of handling in synthesis, a more favorable biodistribution, and lower immunogenicity and have been tested in preliminary clinical experiences.