Journal of Molecular Diagnostics最新文献

The value of human papillomavirus (HPV) testing for cervical cancer screening is well established; however, its use as a primary screening option or as a reflex test after atypical cytology results is now gaining wider acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management. We developed a new analysis method for the RIATOL quantitative PCR assay, and validated and implemented it in the laboratory of clinical molecular pathology at Algemeen Medisch Laboratorium (AML), under national accreditation and following the International Organization for Standardization guidelines. This study presents the successful validation of a high-throughput, multitarget HPV analysis method, with enhanced accuracy on both qualitative and quantitative end results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence. Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources. Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL real-time quantitative PCR assay proved to be a remarkable advancement in high-throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.

Mastocytosis is a heterogeneous disorder characterized by abnormal mast cell accumulation, in which the clinical severity may be explained by distinct molecular mechanisms. This study aimed to explore plasma protein biomarkers associated with systemic mastocytosis subtypes, as well as the cellular origin of the identified proteins. Plasma samples from patients with mastocytosis, including cutaneous mastocytosis (CM), indolent systemic mastocytosis (ISM), and advanced systemic mastocytosis (AdvSM), and a reference group of patients with polycythemia vera, were analyzed by Proximity Extension Assay technology targeting 275 proteins. Furthermore, potential cellular origin was explored using an available single-cell RNA-sequencing data set generated from patients with ISM. The study cohort included 16 patients with CM, 92 patients with systemic mastocytosis (ISM, n = 80; AdvSM, n = 12), and 60 patients with polycythemia vera. A principal component analysis based on 275 plasma proteins revealed one cluster of patients with CM and ISM that was separated from patients with AdvSM. Up to 29 proteins were associated with distinct severe activity in patients with systemic mastocytosis (ISM versus AdvSM), including IL-1 receptor type 1 (IL-1RT1) and tumor necrosis factor ligand superfamily member 13B (TNFSF13B) (q < 0.01). Furthermore, single-cell RNA-sequencing analysis from ISM-derived bone marrow cells revealed that the mRNA for the identified proteins was not exclusive of mast cells. Distinct plasma protein profiles show potential to refine ISM and AdvSM diagnoses, possibly reflecting differences in pathogenic mechanisms and diverse clinical manifestations.

Genetic analysis of congenital adrenal hyperplasia (CAH) has been challenging because of high homology between CYP21A2 and its pseudogene CYP21A1P. This study aimed to evaluate the clinical utility of long-read sequencing (LRS) in diagnosis of CAH attributable to 21-hydroxylase deficiency by comparing with multiplex ligation-dependent probe amplification plus Sanger sequencing. In this retrospective study, 69 samples, including 49 probands from 47 families with high-risk of CAH, were enrolled and blindly subjected to detection of CAH by LRS. The genotype results were compared with control methods, and discordant samples were validated by additional Sanger sequencing. LRS successfully identified biallelic variants of CYP21A2 in the 39 probands diagnosed as having CAH. The remaining 10 probands were not patients with CAH. Additionally, LRS directly identified two pathogenic single-nucleotide variations (SNVs; c.293-13C/A>G and c.955C>T) in the presence of interference caused by nearby insertions/deletions (indels). The cis-trans configuration of two or more SNVs and indels identified in 18 samples was directly determined by LRS without family analysis. Eight CYP21A1P/A2 or TNXA/B deletion chimeras, composed of five subtypes, were identified; and the junction sites were precisely determined. Moreover, LRS determined the exact genotype in two probands who had three heterozygous SNVs/indels and duplication, which could not be clarified by control methods. These findings highlight that LRS could assist in more accurate genotype imputation and more precise CAH diagnosis.

Bloodstream infection is a major cause of morbidity and death worldwide. Timely and appropriate treatment can reduce mortality among critically ill patients. Current diagnostic methods are too slow to inform precise antibiotic choice, leading to the prescription of empirical antibiotics, which may fail to cover the resistance profile of the pathogen, risking poor patient outcomes. Additionally, overuse of broad-spectrum antibiotics may lead to more resistant organisms, putting further pressure on the dwindling pipeline of antibiotics, and risk transmission of these resistant organisms in the health care environment. Therefore, rapid diagnostics are urgently required to better inform antibiotic choice early in the course of treatment. Sequencing offers great promise in reducing time to microbiological diagnosis; however, the amount of host DNA compared with the pathogen in patient samples presents a significant obstacle. Various host-depletion and bacterial-enrichment strategies have been used in samples, such as saliva, urine, or tissue. However, these methods have yet to be collectively integrated and/or extensively explored for rapid bloodstream infection diagnosis. Although most of these workflows possess individual strengths, their lack of analytical/clinical sensitivity and/or comprehensiveness demands additional improvements or synergistic application. This review provides a distinctive classification system for various methods based on their working principles to guide future research, and discusses their strengths and limitations and explores potential avenues for improvement to assist the reader in workflow selection.

Replication-coupled gene editing using locked nucleic acid–modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine–resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.

PMS2 is one of the DNA-mismatch repair genes included in routine genetic testing for Lynch syndrome and colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics' List of Secondary Findings Genes in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read–based next-generation sequencing technologies is complicated by the presence of the pseudogene PMS2CL, and is often supplemented by long-range–based approaches, such as long-range PCR or long-read–based next-generation sequencing, which increases the complexity and cost. This article describes a bioinformatics homology triage workflow that can eliminate the need for long-read–based testing for PMS2 in the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost.

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.

In precision oncology, reliable testing of predictive molecular biomarkers is a prerequisite for optimal patient treatment. Interlaboratory comparisons are a crucial tool to verify diagnostic performance and reproducibility of one's approach. Herein is described the design and results of the first recurrent, internationally performed PIK3CA (phosphatidylinositol-4,5-bisphosphate 3 kinase catalytic subunit α) breast cancer tissue external quality assessment (EQA), organized by German Quality in Pathology GmbH and started in 2021. After the internal pretesting phase performed by the (lead) panel institutes, in both 2021 and 2022, each EQA test set comprised n = 10 tissue samples of hormone receptor–positive, human epidermal growth factor receptor 2–negative invasive breast cancer that had to be analyzed and reported by the participants. In 2021, the results were evaluated separately for German-speaking countries (part 1) and international laboratories (part 2). In 2022, the EQA was performed across the European Union. The EQA success rates were 84.6% (n = 11/13), 88.6% (n = 39/44), and 87.9% (n = 29/33) for EQA 2021 part 1, part 2, and EQA 2022, respectively. The most commonly used methods were next-generation sequencing and mutation-/allele-specific qualitative PCR-based assays. In summary, this recurrent PIK3CA EQA proved to be a suitable approach to obtain an international overview of methods used for PIK3CA mutation analysis, to evaluate them qualitatively, and identify the strengths and weaknesses of individual methods.

Identification of human leukocyte antigen B27 (HLA-B27) by flow cytometry (FCM) has been widely applied in clinical practice for auxiliary diagnosis of ankylosing spondylitis (AS). However, FCM requires freshly prepared samples and relies on expensive equipment, reagents, and an experienced operator. To provide a cheaper and more convenient method for HLA-B27 detection, we proposed a new method termed sequence-encoded fluorescence amplification assay (SEFA), which specially recognized sequences of HLA-B27 gene (HLA-B∗27) covering current common subtypes in a single closed tube. SEFA could detect as low as 10 pg (equal to 3 copies) genomic DNA per reaction and distinguish HLA-B∗27 from other HLA-B alleles with highly similar sequences. A total of 288 clinical samples were tested by SEFA, including 181 patients with AS and 107 healthy controls. Compared with the detection results from FCM, two controversial samples of patients with AS were obtained and further confirmed to be consistent with SEFA by Sanger sequencing, indicating that this method was more accurate than FCM. Moreover, SEFA could detect HLA-B27 status by using supernatant from crude extract of 10-μL blood without commercial reagents. Overall, SEFA has the potential to be an alternative for HLA-B27 identification with the advantage of convenience and low cost, especially suitable for early diagnosis of AS in areas with limited medical resources.