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A novel multi-site enzymatic repairing amplification strategy is developed for high-sensitive terminal deoxynucleotidyl transferase quantification through combining enzymatic repairing amplification and lesion base-involved terminal extension. This method may provide a sensitive and flexible tool for molecular diagnosis and drug discovery. [ABSTRACT FROM AUTHOR]

Monitoring gastrointestinal helminth infections in wild ruminants poses significant challenges for managing wildlife health, particularly regarding invasive species. Traditional coprological methods are often limited by their labor-intensive nature and potential for erroneous identification due to morphological similarities among parasite species. This study employed advanced molecular techniques to assess the prevalence and distribution of several helminth taxa, including the invasive nematode Ashworthius sidemi and the trematode Fascioloides magna , in wild ruminant populations in the Czech Republic (CR). A comprehensive and extensive survey on parasite occurrence, unique in its nationwide scope, was conducted on 983 fecal samples collected from red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), and mouflon (Ovis musimon) across various regions of the CR. The samples were analyzed using multiplex real-time PCR assays specifically designed to detect the DNA of six helminth representatives: the nematodes A. sidemi and Haemonchus spp., as well as the trematodes F. magna , Dicrocoelium dendriticum , Fasciola hepatica , and Calicophoron daubneyi (and representatives of the family Paramphistomidae, respectively). These assays targeted regions of ribosomal DNA (rDNA) and were designed to exhibit high sensitivity and specificity, enabling accurate detection of helminth parasites directly in fecal samples. The molecular assays revealed that invasive nematode A. sidemi was the most prevalent helminth species, detected in 15.8% of all samples (155/983), with the highest infection rate observed in red deer at 30.7% (124/404). Haemonchus spp. were also frequently detected, identified in 14.9% of samples (146/983), particularly in roe deer, with a prevalence of 23.2% (86/371). Spatial analysis of these nematodes across various regions of the CR revealed the extensive distribution of both A. sidemi and Haemonchus spp. in nearly all regions. In contrast, trematode infections were less common, with F. magna and D. dendriticum each found in only 1.5% of samples (15/983). Members of the family Paramphistomidae were detected in 0.2% of the samples (2/983) and were confirmed through sequencing as C. daubneyi. The geographical distribution patterns identified in this study indicate potential hotspots for specific helminth species. These findings are critical for planning health management and conservation strategies to mitigate the impacts of helminth infections, especially in areas affected by invasive species. [ABSTRACT FROM AUTHOR]

Glycosylation, characterized by its complexity and diversity, is a common system across all domains of life. The glycosylation of proteins or lipids imparts them with structural and functional roles, ranging from development to infectious or Mendelian disease. The high-throughput-based omics data has revealed that glycans are involved in important cellular processes. Comprehensive knowledge of glycosylation has contributed not only to the fundamental concepts in glycoscience but also to its applications, including the development of molecular markers for diagnosis and therapeutic tools for treating diseases. The GlyCosmos Glycoscience Portal (GlyCosmos) has undergone significant updates to better support the scientific community in studying glycosylation-related phenomena. Key enhancements include the integration of expanded datasets linking glycans to other omics fields, improved tools for glycan structure prediction and analysis, and upgraded visualization capabilities to streamline data interpretation. A strengthened focus on data standardization has also been introduced, fostering interoperability between glycoscience resources and external databases. Since its release in 2019, the portal has seen a fivefold increase in user engagement, reflecting its growing relevance. These recent advancements aim to provide researchers with a more comprehensive and user-friendly platform, enabling deeper insights into glycan roles in cellular processes and disease mechanisms. GlyCosmos will continue to evolve, prioritizing community needs and advancing the integration of glycoscience with broader biological and biomedical research. [ABSTRACT FROM AUTHOR]

Introduction: Rubinstein–Taybi syndrome (RSTS) is one of the many forms of syndromic intellectual disability, occurring in the population with a frequency of 1: 100–125 thousand newborns. The specific phenotype of patients enables the so-called "portrait" diagnosis of classical cases of RSTS, followed by the analysis of the CREBBP and EP300 genes, whose association with RSTS has been confirmed. Nevertheless, for approximately half of the patients in various cohorts, the diagnosis cannot be confirmed. Methods: In this paper we present the results of a study of 158 Russian patients referred for molecular diagnosis of RSTS using multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS). Results: Pathogenic and likely pathogenic variants were identified in 67 patients (42.4%), of which 62 (39%) were in CREBBP and 4 cases (2%)—in EP300. In one case, a known pathogenic variant in SRCAP , associated with Floating–Harbor syndrome (FHS), which is phenotypically similar to RSTS, was also identified; therefore, the possibilities and prospects for differential diagnosis were considered. [ABSTRACT FROM AUTHOR]

Background: Hereditary spherocytosis (HS) is a group of genetically heterogeneous hereditary hemolytic disorders characterized by anemia, splenomegaly, jaundice, reticulocytosis, and spherical red blood cells on peripheral blood smears. Mutations in key genes, including SPTB , ANK1 , SLC4A1 , SPTA1 , and EPB42 , are commonly implicated in HS. Case Presentation: We report the case of a 22-year-old female presenting with anemia, jaundice, and a family history of splenectomy. Laboratory investigations revealed hemolytic anemia, elevated bilirubin levels, and peripheral blood smear findings consistent with HS. Genetic testing identified a novel SPTB gene splicing mutation (NM_001355436.2: c.1645-1G>A), inherited maternally, which is predicted to disrupt normal RNA splicing and protein synthesis. Discussion: The identified SPTB mutation expands the known mutation spectrum of the SPTB gene and highlights its role in the pathogenesis of HS. Clinical findings, combined with genetic analysis, confirmed the diagnosis of HS and underscored the importance of comprehensive molecular testing for accurate diagnosis, especially in patients with a strong family history. Conclusion: This case emphasizes the utility of genetic testing in diagnosing hereditary spherocytosis, particularly for novel gene mutations. Early and accurate molecular diagnosis facilitates better clinical management, family counseling, and treatment decisions for patients with HS. [ABSTRACT FROM AUTHOR]

Introduction: Cutaneous leishmaniases (CL), a wide range of cutaneous diseases caused by diverse species of Leishmania genus parasites, are among the most neglected infectious diseases. While they are non-fatal, CL are highly morbid with disfiguring lesions, which could be chronic, leaving lifelong unsightly scars; they are combined with psychological distress and social stigma. The efficiency of treatment highly depends on the infecting Leishmania species. Diagnosis is mainly based on microscopic direct examination (DE) of Giemsa-stained smears needing experienced microscopists. It can be laborious and time-consuming when the parasite load is low. DE is poorly sensitive and does not identify Leishmania species. So far, only DNA assays accurately identify the species. Despite their wide use for generic detection, PCR methods also require equipment and additional steps to identify causal Leishmania species. L. major is hyperendemic in many countries in Africa, the Middle East, and Asia, where other species co-occur with different endemicity levels according to the situations. This complicates disease management and treatment, particularly as distribution and epidemiology of leishmaniases remain poorly understood. Here, we aimed for a simple and rapid molecular diagnostic test to detect and identify L. major , a predominant CL causal species, which could be prone to become a control tool at the point of care, in endemic areas, using isothermal recombinase DNA amplification (recombinase polymerase amplification, RPA, or recombinase aided amplification, RAA) coupled to detection by the lateral flow (LF) chromatography on a PCRD cassette. Methods: To develop an L. major species-specific RPA-LF assay, computational analysis of 70 Leishmania DNA targets, identified through bibliography and database searches, selected five targets. We designed and tested 7 primer pairs/probe sets to specifically amplify L. major DNAs. First, the primers were tested for species specificity and sensitivity using basic RPA chemistry. Then, to develop RPA-coupled LF detection, we shifted to the nfo chemistry. Results: This way, we retained one set for further investigation, which confirmed it is L. major species-specific. Tested on 86 human cutaneous samples, this selected set was able to detect 100% of L. major infections in confirmed CL patients. We did not observe any cross-reactivity with lesions due to L. infantum or L. tropica. [ABSTRACT FROM AUTHOR]

Variants in genes encoding for voltage-gated K+ (Kv) channels are frequent cause of drug-resistant pediatric epilepsies. Obtaining a molecular diagnosis gives the opportunity to assess the efficacy of pharmacological strategies based on in vitro features of mutant channels. In this retrospective observational study, we selected patients with drug-resistant pediatric epilepsies caused by variants in potassium channel encoding genes, followed at the Fondazione IRCCS Istituto Neurologico Carlo Besta of Milan, Italy. After the experimental characterization of variants' functional properties in transiently transfected Chinese Hamster Ovary (CHO) cells, we identified drugs to be used as pharmacological approaches. We recruited six patients carrying different missense variants in four Kv channels (Kv7.2, Kv7.3, Kv3.1, and KNa1.1). In vitro experiments demonstrated that variants in Kv7 channels induced loss-of-function (LoF) effects, while those affecting Kv3.1 or KNa1.1 led to gain-of-function (GoF). Moreover, we found that the Kv7 channels activator gabapentin was able to revert the LoF effects caused by Kv7.2/Kv7.3 variants, and the potassium channel-blocker fluoxetine counteracted the GoF effects in Kv3.1 or KNa1.1 variants. According to experimental data, patients carrying Kv7 variants were treated with gabapentin. While this treatment resulted successful in two patients (#1, Kv7.2 G310S variant; #3, Kv7.3 V359L + Kv7.3 D542N), it resulted detrimental in the remaining case (#2, Kv7.2 D535E), requiring drug withdrawal. The application in vivo of fluoxetine to counteract GoF effects induced by Kv3.1 or KNa1.1 variants determined a significant reduction of both seizure frequency and behavior disturbances in patient #4 (Kv3.1 V425M), and in both subjects carrying KNa1.1 variants (#5, S937G and #6, R262Q). However, for the latter case, this drug was halted due to severe behavioral side effects. For most of the patients herein reported, pharmacological strategies, selected according to the in vitro functional properties of Kv-channels pathogenic variants, resulted in a significant improvement of both epileptic and cognitive features. [ABSTRACT FROM AUTHOR]

Common metastasis sites for cervical cancer are the lungs, bones, liver, brain, ovaries, and lymph nodes, among other sites. Skin metastasis is very uncommon, and is only observed in approximately 1% of patients. The cancer spreads typically through lymphatic or blood vessels, but a definitive example of lymphatic spread has not been documented thoroughly in the existing literature. Cutaneous metastasis may be confused with cellulitis or a rash; hence, an immediate cutaneous biopsy of any suspicious lesions is recommended. There is no consensus regarding the treatment of this condition. Only one documented case has shown that a combination of paclitaxel, carboplatin, bevacizumab, and zoledronic acid can lead to a complete metabolic response. Our study, which used two cycles of albumin-bound paclitaxel, cisplatin, and bevacizumab, followed by four cycles of the same regimen plus terprelimab for metastases with CPS scores of Programmed death-ligand 1 (PD-L1) over 10, resulted in a stable complete response for over eight months. Our contribution may assist in formulating effective treatment guidelines for the cutaneous metastasis of squamous cervical carcinoma in the future. [ABSTRACT FROM AUTHOR]

Purpose: Orbital/periorbital tuberculosis (TB) is an uncommon manifestation of extrapulmonary TB, presenting diagnostic challenges due to its varied clinical features that can mimic other diseases. This report aims to present a rare case of periorbital TB in a young man. Methods: Case report. Results: A 36-year-old man presented with painless left periorbital swelling and discharge following facial trauma. Despite initial treatment with antibiotics elsewhere, symptoms persisted. Imaging revealed a periorbital abscess with adjacent bone involvement. Microbiologic studies demonstrated Mycobacterium tuberculosis complex growth, confirming TB. There was no evidence of systemic TB. The patient received anti-tuberculosis therapy. At the 8-month follow-up, he remained symptom-free. Conclusion: Orbital/periorbital TB, although rare, should be taken into consideration in regions with high TB prevalence when dealing with chronic, non-resolving periorbital lesions. Advanced imaging and molecular diagnostics play crucial roles in confirming the diagnosis, especially given the low sensitivity of traditional culture methods for extrapulmonary TB. [ABSTRACT FROM AUTHOR]

Mesophilic microbial sources of prokaryotic Argonaute (pAgo) programmable nucleases have garnered considerable attention for their potential applications in genome editing and molecular diagnostics. In this study, we characterized a novel pAgo from the mesophilic bacterium Chroococcidiopsis sp. (ChAgo), which can cleave single-stranded DNA (ssDNA) using both 5′-phosphorylated guide DNA (5′P-gDNA) and 5′-hydroxylated guide DNA (5′OH-gDNA). Efficient cleavage occurs using 14–25 nt 5′P-gDNA and 13–20 nt 5′OH-gDNA in the presence of Mn2+ ions at temperatures ranging from 25 to 75 °C, with optimal activity at 55 °C. ChAgo demonstrates low tolerance for single-base mismatches, similar to other pAgo proteins. The cleavage efficiency varies based on the guide/target pair, with mismatches at specific positions significantly reducing activity. For instance, mismatches at positions 4, 5, or 12 in T-gDNA/target pairs and at positions 5 or 8–10 in g38NT-gDNA/target pairs notably decrease efficiency. ChAgo's sensitivity to mismatches makes it a promising tool for nucleic acid manipulation and detection, requiring initial screening for high cleavage efficiency sites and subsequent identification of mismatch positions. [ABSTRACT FROM AUTHOR]

Melanoma is the fifth most common cancer in the United States. The advent of immunotherapy and molecular targeted therapy has improved progression-free and overall survival in many patients with advanced disease. However, the selection of therapeutic choices requires a nuanced approach, especially when considering molecularly targeted agents. This case report highlights a diagnostic and therapeutic challenge in managing a patient with a history of chronic lymphocytic leukemia (CLL) and recurrent melanoma. Molecular testing suggested discordant BRAF V600E testing and a simultaneous NRAS G12D mutation. After a careful literature review, repetition of his molecular testing, and analysis of the timelines and results of all his molecular testing, we concluded that the BRAF V600E mutation result was falsely positive. The patient was treated with two cycles of ipilimumab (1 mg/kg) and nivolumab (3 mg/kg) as per the NADINA trial and had a complete radiographic response. He then underwent resection demonstrating a pathologic partial response ranging from 20% to 95% tumor necrosis, dependent on the satellite examined. This case report underscores the importance of precise molecular diagnostics in guiding melanoma treatment and demonstrates the complexities of managing a patient with a coexisting malignancy. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by Trypanosoma cruzi, remains a significant global health challenge, particularly in the molecular diagnostics of low parasitemia during the chronic phase. This highlights the critical need for enhanced diagnostic methodologies. In response, this study evaluates the effectiveness of an automated magnetic beads-based DNA extraction method in improving the molecular diagnosis of Chagas disease compared to the traditional silica column-based extraction. Accordingly, this research seeks to enhance the DNA yield, purity, and sensitivity of real-time PCR (qPCR) assays for detecting T. cruzi satDNA. Blood samples spiked with guanidine–EDTA solution and varying concentrations of T. cruzi were used to compare the two extraction methods. The results indicated that the magnetic bead-based method outperformed the silica column in terms of DNA concentration, purity, and earlier detection of T. cruzi satDNA. Although both methods had similar limits of detection at a 95% confidence interval, the magnetic bead-based approach demonstrated higher sensitivity and reproducibility, particularly in low-parasitemia samples. The findings suggest that the magnetic beads-based DNA extraction method offers a more reliable, faster, and more sensitive alternative for diagnosing chronic Chagas disease, potentially improving clinical outcomes by enabling more accurate and earlier parasite detection. [ABSTRACT FROM AUTHOR]

Summary: The hop cyst nematode, Heterodera humuli , is a pest of hop with the potential to substantially limit yields worldwide. To enable molecular-based diagnostics, a real-time PCR assay for the identification of H. humuli was designed. Polymorphism in the COIII gene fragments was used to design species-specific primers and a TaqMan probe. The specificity of the primer and probe set was tested against different populations of H. humuli and non-target cyst nematodes in multiplex reactions. In multiplex real-time PCR experiments with specific and universal primer and probe sets, fluorescent signals were simultaneously monitored for COIII and D3 of 28S rRNA target genes. Assays with species-specific primers and probe were able to detect DNA extracted from 0.0016 of a H. humuli second-stage juvenile per reaction while showing no reaction with non-target species. [ABSTRACT FROM AUTHOR]

Simple Summary: Equine piroplasmosis is a tick-borne disease with significant health and economic impacts on the equine industry. Various piroplasm species and genotypes belonging to Babesia and Theileria genera have been identified as causative agents of this disease in horses, and recent studies highlight treatment differences depending on the species involved. Therefore, knowing which piroplasm species are circulating in a specific area is crucial, and highly sensitive diagnostic methods are needed to identify and differentiate the pathogen responsible for the infection. The aims of our study were to compare the diagnostic performance of different molecular tests for piroplasm DNA detection and genetically characterize the piroplasms identified in 63 horses in Northern Italy from 2016 to 2022. Molecular analysis revealed a 38.1% positivity rate within the tested population. Notably, substantial genetic variability was observed among the identified theileria rather than among the babesia. No single diagnostic method was found to reliably detect and differentiate all piroplasm species involved in equine piroplasmosis. This study highlights the need for further investigation into the genetic diversity of these parasites. Expanding our understanding of piroplasm variability is essential to develop and implement appropriate diagnostic methods for the accurate detection of equine piroplasmosis. Equine piroplasmosis is a tick-borne disease caused by Babesia and Theileria species. Despite its presence in Europe, no laboratory testing is required for animal movement, even though some countries remain free of this disease. Differentiating between species and genotypes is crucial to determine the most effective treatment, as dosage, active compounds, and duration vary. However, diagnosis is often challenging due to genetic variability and the limited sensitivity of molecular methods. The aims of this study were to compare the performances of different molecular diagnostic tests to identify the most effective assay for piroplasm DNA detection and to genetically characterize the piroplasms identified in horses in Northern Italy from 2016 to 2022. Among 63 horses tested, 24 (38.1%) were positive in at least one of the tests used. Four horses tested positive for Babesia caballi with identical nucleotide sequences, while 22 horses tested positive for genetically different Theileria species, including Theileria equi, Theileria haneyi-like species, and Theileria sp. Africa. Two horses were coinfected by Babesia caballi and Theileria haneyi-like species. The best diagnostic approach to avoid false negative results was a combination of different assays. Further studies will be necessary to better assess the prevalence and genetic diversity of piroplasms involved in equine piroplasmosis. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) complicated by disseminated intravascular coagulation (DIC) poses major diagnostic and therapeutic challenges. While DIC is well documented in acute promyelocytic leukemia, its manifestations in non-APL AML remain underexplored, necessitating precise diagnostic strategies for effective management. Methods: AML patients with overt DIC were analyzed, including morphological, immunophenotypic, cytogenetic, and genetic evaluations. DIC was diagnosed using the ISTH scoring system, and AML subtypes were classified following WHO criteria. Results: Three diagnostic patterns were identified. (1) Acute promyelocytic leukemia: Leukemia characterized by PML::RARa rearrangements, FLT3 co-mutations, and frequent Auer rods and faggot bundles. Immunocytological analysis showed CD34 and HLA-DR negativity. (2) AML with FLT3 and/or NPM1 mutations: A high prevalence of cup-like blasts was found in 70% of cases. FLT3 mutations, often co-occurring with NPM1, dominated, while karyotypes were typically normal. Immunophenotyping revealed strong myeloid marker expression (MPO+, CD13+, and CD33+), with occasional CD34 negativity. (3) AML with monocytic differentiation: Leukemia defined by monoblastic/promonocytic morphology, DNMT3A mutations, and complex karyotypes or 11q23 rearrangements. Immunophenotyping demonstrated a dominance of monocytic markers (CD4+, CD14+, CD15+, and CD64+). Two patients presented unique profiles with no alignment to these patterns. Conclusions: This study highlights distinct hematopathological patterns of AML with overt DIC, providing a framework for early and precise diagnosis. Recognizing these patterns is critical for tailoring diagnostic and therapeutic approaches to improve outcomes in this high-risk population. [ABSTRACT FROM AUTHOR]

Background/Objectives: The accurate detection of EGFR mutations, particularly the rare S768I variant, is crucial for guiding treatment decisions in non-small cell lung cancer (NSCLC) patients. This study investigated the incidence of false positives in S768I mutation detection using the IdyllaTM qPCR system and compared results with next-generation sequencing (NGS). Methods: A prospective observational study was conducted at the Dr. Negrín University Hospital between July 2023 and July 2024. Six NSCLC patient samples with S768I variant detection by IdyllaTM were analyzed from all NSCLC cases tested during the study period. Initial testing was performed on tissue samples (Idylla1), followed by replicate analysis using extracted DNA (Idylla2). Results were compared with NGS as the reference method. Statistical analysis included the calculation of sensitivity, specificity, accuracy, and Kappa concordance index. Results: Initial Idylla testing showed an 80% false positive rate, with only one of five positive results confirmed by NGS. The first analysis demonstrated high sensitivity (100%) but low specificity (20%), with an accuracy of 0.333 and poor concordance with NGS (Kappa = 0.077). Repeat testing using extracted DNA showed improved performance, with increased accuracy (0.833) and better agreement with NGS (Kappa = 0.571). Analysis of amplification curves revealed that false positives typically showed normalized fluorescence values below 12 points, with no clear correlation between false positives and factors such as sample quantity or tumor content. Conclusions: While the IdyllaTM system shows high sensitivity for S768I detection, its initial specificity is problematic, leading to frequent false positives. These findings emphasize the importance of confirming positive S768I results through alternative methods like NGS, particularly when these results could influence therapeutic decisions. Results suggest the need to refine the system's interpretation algorithms to improve specificity. [ABSTRACT FROM AUTHOR]

Background Gorlin syndrome (GS) is an autosomal dominant disorder characterized by a predisposition to basal cell carcinoma and developmental defects. It is caused by pathogenic variants in the PTCH1 or SUFU genes. Objectives To ascertain the effectiveness of molecular screening in a cohort of patients with a suspicion of GS and to describe the patients' clinical and genetic characteristics. Methods In total, 110 patients with a suspicion of GS were studied. The patients were seen at the genetic department of Bichat University Hospital for molecular screening. The patients' clinical and paraclinical data were collected and analysed according to Evans' diagnostic criteria and were compared with molecular information. Results Among 110 probands, only 56% fulfilled Evans' diagnostic criteria. Overall, 75% of the patients who fulfilled those criteria carried a pathogenic variation in PTCH1 or SUFU. We compared the clinical and paraclinical data of 54 probands carrying a PTCH1 or SUFU mutation with 56 probands without identified mutations. Among patients carrying a pathogenic variation in the PTCH1 or SUFU genes, 30 years appears to be the cut-off age after which all patients have clear clinical GS. Indeed, after age 30 years, all patients carrying a PTCH1 or SUFU mutation fulfilled the diagnostic criteria of Evans (82% met the clinical criteria, reaching 100% with complementary examinations such as X-rays and ultrasound). Before 30 years of age, only 37% of patients with mutated genes fulfilled the clinical diagnostic criteria, reaching only 62% with simple complementary exams. We also report 22 new mutations in PTCH1. Conclusions Molecular screening of patients with GS who do not fulfil Evans' diagnostic criteria should only be offered in the first instance to patients under 30 years of age. After age 30 years, careful clinical examination and complementary radiological exams should be enough to eliminate the diagnosis of GS among patients who do not fulfil the diagnostic criteria. [ABSTRACT FROM AUTHOR]

Objective: This study aims to evaluate our experience in the diagnosis of hereditary ataxias (HAs), to analyze data from a real-world scenario. Study design: This is a retrospective, cross-sectional, descriptive study conducted at a single Italian adult neurogenetic outpatient clinic, in 147 patients affected by ataxia with a suspicion of hereditary forms, recruited from November 1999 to February 2024. A stepwise approach for molecular diagnostics was applied: targeted gene panel (TP) next-generation sequencing (NGS) and/or clinical exome sequencing (CES) were performed in the case of inconclusive first-line genetic testing, such as short tandem repeat expansions (TREs) testing for most common spinocerebellar ataxias (SCA1-3, 6–8,12,17, DRPLA), other forms [Fragile X-associated tremor/ataxia syndrome (FXTAS), Friedreich ataxia (FRDA) and mitochondrial DNA-related ataxia, RFC1-related ataxia/CANVAS] or inconclusive phenotype-guided specific single gene sequencing. Result: A definitive diagnosis was reached in 36.7% of the cases. TREs testing was diagnostic in 30.4% of patients. The three most common TREs ataxias were FRDA (36.1%), SCA2 (27.8%), and RFC1-related ataxia/CANVAS (11.1%). In five patients, the molecular diagnosis was achieved by single gene sequencing and causative mutations were identified in POLG (2), SACS (1), DARS2 (1), MT-ATP6 (1). Of 94 patients with a suspicion of HAs of indeterminate genetic origin, 68 underwent new molecular evaluation using the NGS approach. In 28 of these cases, CES was performed after the TP sequencing resulted negative. In 13 patients, the diagnosis was achieved by NGS approach. In 7 of these 13 patients, the diagnosis was made by CES. Genes mutations identified as causative of HAs were found in SPG7 (4), SACS (1), CACNA1A (1), CACNA1G (1), EEF2 (1), PRKCG (1), KCNC3 (1), ADCK3 (1), SYNE1 (1), ITPR1 (1). A positive family history of ataxia and early onset of symptoms were associated with a higher likelihood of obtaining a definite diagnosis. Conclusion: The molecular diagnosis of HAs remains a significant challenge for neurologists. Our data indicate that, in most cases, a diagnosis of HA can be established through first line genetic testing, particularly TREs testing. However, for patients with a clinical diagnosis of HA who do not achieve a molecular diagnosis through initial genetic tests, the use of NGS proves to be a valuable tool, providing a definitive diagnosis in approximately 20% of cases. Therefore, when feasible in clinical practice, integrating NGS testing, especially exome sequencing, into the diagnostic decision-making process for unsolved cases is crucial. [ABSTRACT FROM AUTHOR]

Purpose and methods: This prospective study aims to diagnose the etiology of non-focalized fever lasting between 5 and 28 days in the islands of La Palma and El Hierro (Canary Islands, Spain) during 2021, using serology and PCR. Results: The etiological profile described in this study aligns with that of fever of intermediate duration (FID), with zoonoses being the primary cause. Murine typhus (MT) is identified as the leading cause, followed by Q fever (QF). The incidence of MT is the highest reported nationally and comparable to the highest in Europe, with 39.6 cases per 100,000 inhabitants in La Palma and 79.7 cases per 100,000 inhabitants in El Hierro. Q fever, known to be endemic to the Canary Islands, presents incidences of 26.5 cases per 100,000 inhabitants in La Palma and 15.6 cases per 100,000 inhabitants in El Hierro. MT shows no gender differences and has a homogeneous geographical distribution. In contrast, QF is more prevalent in men and has a heterogeneous geographical distribution. Conclusions: The high incidence of MT found in both urban and peri-urban areas is particularly noteworthy. Its potential connection with climate change and/or the growth of the reservoir population in the Canary Islands remains unknown. MT's similarity to QF in terms of clinical signs and treatment, coupled with the absence of a specific protocol for early diagnosis, may have contributed to its underdiagnosis. MT can lead to significant health concerns, including risk of hospitalization, complications, and even death. Therefore, the registration of cases for epidemiological control is deemed essential. [ABSTRACT FROM AUTHOR]

Background: Meniscal injuries that fail to heal instigate catabolic changes in the knee's microenvironment, posing a high risk for developing posttraumatic osteoarthritis (PTOA). Previous research has suggested that human cartilage–derived progenitor cells (hCPCs) can stimulate meniscal repair in a manner that depends on stromal cell–derived factor 1 (SDF-1) pathway activity. Hypothesis: Overexpressing the SDF-1 receptor CXCR4 in hCPCs will increase cell trafficking and further improve the repair efficacy of meniscal injuries. Study Design: Controlled laboratory study. Methods: hCPCs were genetically modified to overexpress CXCR4 (CXCR4-overexpressing [OE] hCPCs) using lentivirus. In vitro characterization was performed using cell viability assay, cell migration assay, and immunoblotting. These cells were then used to treat a meniscal injury in rabbits. A medial meniscal tear was surgically created in the right knees of New Zealand White rabbits, followed by 2 intra-articular injections (5.0 × 106 cells each) of either CXCR4-OE hCPCs, wild-type hCPCs, or saline alone. A histological assessment of menisci and cartilage was performed using safranin O/fast green staining. Joints were assessed for PTOA changes using the modified Osteoarthritis Research Society International scoring system. Fluorescence imaging and DNA analysis were performed to examine tissue for human cells. Results: SDF-1 inhibited NF-κB and ERK pathways in both wild-type and CXCR4-OE hCPCs. CXCR4 overexpression increased hCPC trafficking toward sources of SDF-1, including injured meniscal fibrocartilage and an SDF-1–presoaked collagen scaffold. Intra-articular injections of CXCR4-OE hCPCs significantly improved meniscus healing, as evidenced by the complete absence of tears in 5 of 6 (83%) animals that received CXCR4-OE hCPCs compared with only 3 of 6 (50%) wild-type hCPC–treated animals and 2 of 6 (33%) animals in the saline control group. CXCR4-OE hCPC–treated animals also showed significantly less erosion in their knee cartilage compared with control animals. Conclusion: Overall, CXCR4 overexpression inhibited catabolic pathway signaling in hCPCs and increased cell migration. Evidence suggests that intra-articular injections of these cells into the injured knee allow them to home in on sites of fibrocartilage injuries and ultimately result in meniscal tear healing and PTOA inhibition in immunocompetent animals. Clinical Relevance: This study demonstrated that cartilage progenitors with elevated CXCR4 expression have the potential to be a potent therapeutic tool for stimulating meniscal tear healing. [ABSTRACT FROM AUTHOR]

Background Isolated tubulitis, borderline changes and isolated arteritis suspicious for histologic T-cell-mediated rejection (hTCMR) remain findings of uncertain significance. Although the Molecular Microscope Diagnostics System (MMDx) has not been trained on those lesions, it was suggested that MMDx might reclassify a subgroup to molecular TCMR (mTCMR). Methods In this single-center cohort of 326 consecutive, unselected kidney allograft biopsies assessed by histology and MMDx, we analyzed 249 cases with isolated tubulitis (i0, t1–3, v0; n  = 101), borderline changes (according to Banff 2022, v0; n  = 9), isolated arteritis (no borderline, v1; n  = 37), no inflammation (i0, t0, v0; n  = 67) and a positive control cohort (hTCMR, n  = 27; mixed histologic rejection, n  = 8; both according to Banff 2022; total n  = 35). The first three groups were summarized as TCMR-suspicion (n  = 147). Subcategorization included the presence and absence of microvascular inflammation (MVI); g+ptc ptc ≥2. Molecular rejection rates and differentiation were investigated. Results Molecular rejection rates were 37/147 cases (25.2%; 32 with MVI) in TCMR-suspicion, 6/67 (9%; 4 with MVI) in no inflammation and 30/35 (85.7%; 19 with MVI) in the positive control cohort. Molecular antibody-mediated rejection (mAMR) was present in 39/73 (53.4%) of cases. The presence of donor-specific antibodies at the time of the biopsy was high (127/249, 51%). Only 3 mAMR/TCMR and 0 pure mTCMR cases were detected in TCMR-suspicion and no inflammation, compared with 12 mAMR/TCMR and 10 mTCMR cases in the positive control cohort (P  < .001). Even though the TCMR-specific molecular (Classifier) score differentiated between TCMR-suspicion and no inflammation (P  = 0.005), rejection phenotype scores (R2 and R3) did not (P  = .157 and.121). Conclusions MMDx did not identify pure mTCMR among isolated tubulitis, borderline changes or isolated arteritis, likely due to low sensitivity for TCMR lesions. However, it identified mAMR or mAMR/TCMR, especially in cases with MVI. Subthreshold findings remain to be further studied. [ABSTRACT FROM AUTHOR]

Purpose: The emerging zoonotic Borna disease virus 1 (BoDV-1) and the variegated squirrel bornavirus 1 (VSBV-1) cause severe and fatal human encephalitis in Germany. We conducted the first systematic clinical analysis of acute, molecularly confirmed fatal bornavirus encephalitis cases comprising 21 BoDV-1 and four VSBV-1 patients to identify options for better diagnosis and timely treatment. Methods: Analyses were based on medical records and, for BoDV-1, on additional medical interviews with patients' relatives. Results: Disease onset was unspecific, often with fever and headache, inconsistently mixed with early fluctuating neurological symptoms, all rapidly leading to severe encephalopathy and progressive vigilance decline. Very shortly after seeking the first medical advice (median time interval 2 and 0 days for BoDV-1 and VSBV-1, respectively), all except one patient were hospitalised upon manifest neurological symptoms (median 10 and 16 days respectively after general symptom onset). Neurological symptoms varied, always progressing to coma and death. BoDV-1 and VSBV-1 patients required ventilation a median of three and five days, and died a median of 32 and 72 days, after hospitalisation. Death occurred mostly after supportive treatment cessation at different points in time based on poor prognosis. Disease duration therefore showed a wide, incomparable range. Conclusion: The extremely rapid progression is the most obvious clinical characteristic of bornavirus encephalitis and the timeframe for diagnosis and targeted therapy is very short. Therefore, our results demand an early clinical suspicion based on symptomatology, epidemiology, imaging, and laboratory findings, followed by prompt virological testing as a prerequisite for any potentially effective treatment. [ABSTRACT FROM AUTHOR]

Background An eschar is not always present in all scrub typhus patients. Furthermore, such patients may present to tertiary care hospitals after administration of doxycycline. The present study aimed to determine the usefulness of using the swab from eschar sites in the diagnosis of scrub typhus in patients who present post-doxycycline therapy. Methods Blood, eschar scraping and swab samples were collected daily until patient discharge/death. Real-time SYBR green PCR targeting the groEl gene, TaqMan probe PCR targeting the 47 kDa gene and nested PCR targeting the 56 kDa gene of Orientia tsutsugamushi were carried out. Partial sequences of the 56 kDa gene of O. tsutsugamushi were sequenced by the Sanger method and phylogenetic analysis was performed using Mega X. Results In total, 42 samples (19 eschar scraping and 23 eschar swab samples) were collected from 22 patients. A high positivity of eschar scraping samples (89.5%, 17/19) in comparison to blood (63.2%, 12/19) was observed. The nested PCR for eschar swab samples was positive in 10 (43.5%), 47 kDa gene in nine (39.1%) and groEl in three (13%) samples. The swabs remained positive for 1–4 d after doxycycline was started. The majority of the sequences clustered with Karp-like strains. Conclusion The eschar swab is a good alternative sample for the diagnosis and genotyping of scrub typhus. It also has the added advantage of persistent positivity despite doxycycline administration. [ABSTRACT FROM AUTHOR]

Context Genetic testing for 21-hydroxylase deficiency (21-OHD) is always challenging. The current approaches of short-read sequencing and multiplex ligation-dependent probe amplification (MLPA) are insufficient for the detection of chimeric genes or complicated variants from multiple copies. Recently developed long-read sequencing (LRS) can solve this problem. Objective To investigate the clinical utility of LRS in precision diagnosis of 21-OHD. Methods In the cohort of 832 patients with 21-OHD, the current approaches provided the precise molecular diagnosis for 81.7% (680/832) of cases. LRS was performed to solve the remaining 144 cases with complex chimeric variants and 8 cases with variants from multiple copies. Clinical manifestations in patients with continuous deletions of CYP21A 2 extending to TNXB (namely CAH-X) were further evaluated. Results Using LRS in combination with previous genetic test results, a total of 16.9% (281/1664) CYP21A1P/CYP21A2 or TNXA/TNXB chimeric alleles were identified in 832 patients, with CYP21A1P / CYP21A2 accounting for 10.4% and TNXA / TNXB for 6.5%. The top 3 common chimeras were CYP21 CH-1, TNX CH-1, and TNX CH-2, accounting for 77.2% (217/281) of all chimeric alleles. The 8 patients with variants on multiple copies of CYP21A2 were accurately identified with LRS. The prevalence of CAH-X in our cohort was 12.1%, and a high frequency of connective tissue-related symptoms was observed in CAH-X patients. Conclusion LRS can detect all types of CYP21A2 variants, including complex chimeras and pathogenic variants on multiple copies in patients with 21-OHD, which could be utilized as a first-tier routine test for the precision diagnosis and categorization of congenital adrenal hyperplasia. [ABSTRACT FROM AUTHOR]

Background: Gardnerella is a key pathogen in bacterial vaginosis (BV), but the role of the different Gardnerella species remains unclear. We investigated the role of four Gardnerella species, as well as Fannyhessea vaginae , Lactobacillus crispatus and L. iners in BV. Methods: From 331 pregnant women from the Democratic Republic of the Congo, BV was diagnosed using Nugent scoring and a cervicovaginal lavage was used to quantify G. leopoldii , G. piotii , G. swidsinskii , G. vaginalis, F. vaginae, L. crispatus and L. iners by qPCR. Univariate associations between these species and clinical outcomes were assessed. A logistic regression model and ROC curves were calculated to determine the best diagnostic marker for BV. Results: Here, L. iners (75.8%) was the most prevalent species and G. vaginalis (36.0%) the most common Gardnerella species. All investigated Gardnerella spp. were prevalent (50.9-57.9%) in women with (asymptomatic) BV. Univariate analysis revealed no significant associations with clinical symptoms of BV, while F. vaginae (positive Whiff test, high pH), G. vaginalis (high pH) and L. crispatus (low pH) were associated with signs of BV. G. piotii was associated with markers of urinary tract infection. Women with L. iners had higher odds of delivering preterm. ROC analyses showed that F. vaginae was the best marker for BV (AUC 0.81), and the combined model further increased the diagnostic performance (AUC 0.90). Conclusion: All Gardnerella species were involved in BV, although none were associated with the most important clinical symptoms of BV and none emerged as a superior molecular marker for BV. [ABSTRACT FROM AUTHOR]

Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) is challenging because of its depth, which often leads to misdiagnosis during ultrasound examinations. The unique PDAC tumor microenvironment (TME) is characterized by significant fibrous tissue growth, and high interstitial pressure hinders drug penetration into tumors. Additionally, hypoxia and immune suppression within the tumor contribute to poor responses to radiotherapy and chemotherapy, ultimately leading to an unfavorable prognosis. In this study, aPD-L1-modified docetaxel and perfluoropentane-loaded liquid‒vapor phase-transformation lipid nanoparticles (aPDL1-DTX/PFP@Lipid) were synthesized and had an average diameter of 61.63 nm with 84.3% antibody modification. We demonstrated that the nanoparticles (NPs) exhibited excellent PDAC-targeting capabilities both in vitro and in vivo. Upon exposure to low-intensity pulsed ultrasound (LIPUS) stimulation, the NPs underwent a phase transformation to form microbubbles with substantial molecular ultrasound diagnostic effects, and combined treatment resulted in a tumor growth inhibition rate of 88.91%. This treatment strategy also led to the infiltration of CD8+ T cells, the downregulation of Treg cells, the promotion of M1 macrophage polarization, the inhibition of fibrosis to reduce tumor stromal pressure, and the facilitation of perfluoropentane (PFP) gasification to release O2 and improve tumor hypoxia. In conclusion, aPD-L1-modified liquid‒vapor phase-transformation nanoparticles loaded with docetaxel (DTX) and PFP were successfully combined with ultrasound for the molecular diagnosis and targeted treatment of PDAC. aPDL1-DTX/PFP@Lipid could reshape the PDAC TME, offering a new approach for ultrasound-mediated diagnosis and treatment with promising clinical applications. [ABSTRACT FROM AUTHOR]

Introduction: Lupus nephritis (LN) is one of the most frequent and serious organic manifestations of systemic lupus erythematosus (SLE). Autophagy, a new form of programmed cell death, has been implicated in a variety of renal diseases, but the relationship between autophagy and LN remains unelucidated. Methods: We analyzed differentially expressed genes (DEGs) in kidney tissues from 14 LN patients and 7 normal controls using the GSE112943 dataset. Key modules and their contained genes were identified utilizing weighted gene co-expression network analysis (WGCNA). Differentially expressed autophagy-related genes (DE-ARGs) among DEGs, key module genes and autophagy-related genes (ARGs) were obtained by venn plot, and subjected to protein-protein interaction network construction. Two machine learning methods were applied to identify signature genes. The area under the receiver operating characteristic (ROC) curves was used to assess the accuracy of the signature genes. We also analyzed immune cell infiltration in LN. Additionally, the association between key genes and kidney diseases was predicted. Finally, key genes expression in kidney was verified by clinical samples and animal experiments. Results: A total of 10304 DEGs were identified in GSE1129943 and 29 modules were identified in WGCNA. Among them, the brown module and coral 2 module exhibited significant correlation with LN (cor = 0.86, -0.84, p<0.001). Machine learning techniques identified 5 signature genes, but only 2 were validated in the external dataset GSE32591, namely MAP1LC3B (AUC = 0.920) and TNFSF10 (AUC = 0.937), which are involved in autophagy and apoptosis. Immune infiltration analysis suggested that these key genes may be associated with immune cell infiltration in LN. In addition, these genes have been linked to a variety of renal diseases, and their expression was verified in kidney tissues in LN patients and lupus mice. Conclusion: MAP1LC3B and TNFSF10 may be key autophagy-related genes in LN. These key genes have the potential to provide new insights into the molecular diagnosis and treatment of LN. [ABSTRACT FROM AUTHOR]

Long-read sequencing has emerged as a transformative technology in recent years, offering significant potential for the molecular diagnosis of unresolved genetic disorders. Despite its promise, the comprehensive detection and clinical annotation of genomic variants remain intricate and technically demanding. We present SUMMER, an integrated and structured workflow specifically designed to process raw Nanopore sequencing reads. SUMMER facilitates an in-depth analysis of multiple variant types, including SNV, SV, short tandem repeat and mobile element insertion. For clinical applications, SUMMER employs SvAnna to prioritize SV candidates based on phenotype relevance and utilizes Straglr to provide reference distributions of non-pathogenic unit counts for 55 known pathogenic short tandem repeats. By addressing critical challenges in variant detection and annotation, SUMMER seeks to advance the clinical utility of long-read sequencing in diagnostic genomics. SUMMER is available on the web at https://github.com/carolhuaxia/summer. [ABSTRACT FROM AUTHOR]

The development of bispecific antibodies (BsAbs) represents a significant advancement in therapeutic antibody design, enabling the simultaneous targeting of two different antigens. This dual-targeting capability enhances therapeutic efficacy, particularly in complex diseases like cancer, where tumor heterogeneity presents a significant challenge for traditional treatments. By bridging two distinct pathways, BsAbs can improve specificity and minimize off-target effects, making them invaluable in therapeutic contexts. Integrating advanced imaging techniques, particularly Correlative Light and Electron Microscopy (CLEM), offers a unique opportunity to visualize the dynamic interactions of BsAbs within cellular environments. CLEM combines the strengths of optical and electron microscopy, allowing researchers to observe real-time antibody-antigen interactions at nanoscale resolution. This synergy not only deepens our understanding of BsAbs' mechanisms of action but also provides critical insights into their spatial distribution, binding kinetics, and functional dynamics in live cells. In this review, the integration of BsAbs and CLEM paves the way for targeted therapeutic strategies, fostering the development of more effective treatments that can adapt to the complexities of disease pathology. [ABSTRACT FROM AUTHOR]

Introduction: The diagnosis and detection of pathogens such as Rickettsia and Orientia is a cause of major concern among the public health community. Unavailability of rapid, cost-effective diagnostic assays contributes to delayed diagnosis and timely treatment. Using the methodology of systematic reviewing and meta-analysis, the study aimed to synthesize and compare the diagnostic performances of all the available isothermal assays for the detection of classical rickettsial diseases. Methods: Studies were retrieved from PubMed and Scopus, and selection and screening were conducted using pre-determined inclusion and exclusion criteria. Analysis was performed using Meta-DiSc 2.0 for the assessment and comparison of diagnostic performance of the isothermal assays. Results: Overall, six studies were selected as a part of this systematic review. All the selected studies (n = 6) optimized LAMP as their index test to detect scrub typhus. The quality assessment of the selected studies revealed only (n = 1) study to be of poor quality with a QUADAS-2 score of (<2). Meta-analysis revealed the pooled sensitivity of LAMP to be 66% [95% CI (0.40–0.85)] with a pooled specificity of 94% [95% CI (0.81–0.98)]. LAMP was estimated with a positive likelihood ratio of 8.3 [95% CI (3.8–18.1)] and a negative likelihood ratio of 0.3 [95% CI (0.2–0.7)] with a false positivity rate of 0.07 [95% CI (0.02–0.2)]. The diagnostic odds ratio was reported to be 21.96 [95% CI (10.2–47.3)]. Due to severe heterogeneity in the body of evidence (I 2 = 0.77), a meta-regression was performed with certain covariates to explore the potential causes. A case–control design was found to exaggerate the sensitivity {0.84 [95% CI (0.5–0.9)]} and specificity {0.73 [95% CI (0.6–0.8)]}. Conclusion: The findings reveal subpar performance of LAMP for the detection of scrub typhus. Active research and development focused on optimization of novel molecular diagnosis that are efficient, rapid, and cost-effective shall foster timely diagnosis and aid in reduction of the overall burden of scrub typhus. Protocol and registration: A detailed protocol of this review is registered and available in Prospero at: https://www.crd.york.ac.uk/prospero/. (registration number CRD42024511706). [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is an increasingly recognized childhood developmental disorder. Despite extensive study, causal variants and molecular diagnosis remain elusive. There is both heterogeneity of the phenotype, as well as the genetic landscape associated with phenotype, which includes both inherited and de novo mutations. Currently, diagnosis is complex and behaviourally based, oftentimes occurring years after the ideal 1–2 years of age. Structural variants (SVs) are large and sometimes complex genomic variants that are likely underrepresented contributors to ASD due to the limitations of short-read DNA sequencing, such as alignment in repetitive regions and regions with GC bias. Here, we performed long-read sequencing (LRS) on four individuals with autism spectrum disorder to delineate SV complexity and determine precise breakpoints for SVs, which was not possible with short-read whole-genome sequencing (SRS). We use LRS to interrogate the methylation pattern associated with the SVs and phase the SV haplotypes to further clarify their contribution to disorder. LRS allows insight into the genome and methylome that allow us to uncover variant complexity and contribution that was previously unseen with SRS. Ultimately, this furthers precision diagnosis and contributes to individualized treatment for affected individuals and their families within the clinic. [ABSTRACT FROM AUTHOR]

Background: Stereotactic biopsy of posterior fossa lesions, which are often inoperable, enables a safe trajectory and provides tissue samples for accurate diagnosis, which is crucial for correct treatment since the latest World Health Organization Classification of Tumors of the Central Nervous System from 2021 places immense emphasis on molecular diagnostics. Stereotactic biopsy using the Leksell Vantage headframe is, due to its rigid design, extremely accurate, but stiffer, making the procedure more challenging and the learning curve steeper. Methods: This retrospective analysis demonstrates the introduction of the new Leksell Vantage headframe in day-to-day practice at the University Medical Center in Maribor, Slovenia, in demanding procedures of posterior fossa biopsies, and also provides a review of the literature available on the topic with emphasis on the technical aspect of posterior fossa biopsy using the Leksell Vantage headframe in adults. Results: In the observed series of three patients with posterior fossa lesions, all biopsies were representative, despite tissue samples being small, providing conclusive histopathologic reports (glioblastoma, rosette-forming glioneuronal tumor and metastasis of melanoma) with additional molecular diagnostics. After the initial biopsy case, the preoperative planning times and procedure times were shortened as we learnt about the importance of a tailored approach from the first case. In all cases, the biopsy was performed under local anesthesia with patients being awake throughout surgery. Conclusions: The rigid Leksell Vantage headframe makes access to the posterior fossa tougher when compared to its predecessors. However, the procedure is very accurate but requires precise preoperative planning and a customized approach when placing the headframe. [ABSTRACT FROM AUTHOR]

In immunocompromised patients, pneumonia presents a diagnostic challenge due to diverse etiologies, nonspecific symptoms, overlapping radiological presentation, frequent co-infections, and the potential for rapid progression to severe disease. Thus, timely and accurate diagnosis of all pathogens is crucial. This narrative review explores the latest advancements in microbiological diagnostic techniques for pneumonia in immunocompromised patients. It covers major available microbiological tools for diagnosing both community-acquired and hospital-acquired pneumonia, encompassing a wide spectrum of pathogens including bacterial, viral, fungal, and parasitic. While traditional culture methods remain pivotal in identifying many pneumonia-causing etiologies, their limitations in sensitivity and time to results have led to the rise of non-invasive antigen tests and molecular diagnostics. These are increasingly employed alongside cultures and microscopy for more efficient diagnosis, mainly in viral and fungal infections. Lastly, we report the future of pneumonia diagnostics, exploring the potential of metagenomics and CRISPR/Cas13a for more precise and rapid pathogen detection in immunocompromised populations. [ABSTRACT FROM AUTHOR]

Background/Objectives: Candida auris is an emerging multidrug-resistant pathogen with the potential to cause invasive fungal infections and healthcare-associated outbreaks. Currently, there is no systematic review explicitly focusing on the up-to-date molecular diagnostics of this pathogen to cover the entire process, including sample pre-extraction procedures, nucleic acid extraction, and DNA-based detection. Sample pre-treatment and extraction are the prerequisites before molecular testing and have implications on the downstream detection but have not been reviewed elsewhere. This review aims to summarize a comprehensive update in the past 5 years. Methods: A systematic review was conducted to search for articles published in the period between 1 January 2020 and 20 November 2024 from various databases, including PubMed, Google Scholar, and Web of Science. The findings were produced through narrative synthesis, with quantitative analysis conducted where applicable. Results: Starting from 1115 records, 28 studies that met the inclusion criteria were included in the analysis. This review summarized the key updates on three categories, including (i) sample pre-extraction procedures and nucleic acid extraction, including magnetic, bead-beating, mechanical, chemical, thermal, and column-based protocols; (ii) commercial molecular assays; and (iii) laboratory-developed tests (LDTs). For real-time PCR, commercial molecular assays and LDTs showed sensitivity (ranging from 94.9% to 100% and 44% to 100%, respectively) and specificity (ranging from 98.2% to 100% and 92% to 100%, respectively). Conclusions: Here, we describe a useful summary to enlighten readers from clinical microbiology laboratories on the nucleic acid extraction protocols and performance of various molecular diagnostic assays used for the detection of C. auris. [ABSTRACT FROM AUTHOR]