A novel digital PCR assay for detection and comprehensive characterization of Molluscum contagiosum virus genotypes MOCV1, MOCV2, and MOCV3 and recombinant lineages.

Molluscum contagiosum virus (MOCV) is an important human pathogen causing a high disease burden worldwide. It is the last exclusively human-infecting poxvirus still circulating in its natural reservoir-a valuable model of poxviral evolution. Unfortunately, MOCV remains neglected, and little is known about its evolutionary history and circulating genomic variants, especially in non-privileged countries. The design weaknesses of available MOCV detection/genotyping assays surfaced with recent accumulation of abundant sequence information: all existing MOCV assays fail at accurate genotyping and capturing sub-genotype level diversity. Because complete MOCV genome characterization is an expensive and labor-intensive task, it makes sense to prioritize samples for whole-genome sequencing by diversity triage screening. To meet this demand, we developed a novel assay for accurate MOCV detection and genotyping, and comprehensive sub-genotype qualification to the level of phylogenetic groups (PGs). The assay included a novel set of oligonucleotide primers and probes, and it was implemented using digital polymerase chain reaction (dPCR). It offers sensitive, specific, and accurate detection, genotyping (MOCV1-MOCV3), and PG qualification (PG1-6) of MOCV DNA from clinical samples. The novel dPCR assay is suitable for MOCV diversity triage screening and prioritization of samples for complete MOCV genome characterization.

Long-term storage stability of PGL-I coated ELISA plates for anti-Mycobacterium leprae IgM detection.

The assessment of ELISA plates coated with phenolic glycolipid-I/PGL-I revealed excellent stability during eight years of storage at room temperature, promoting consistent IgM antibody detection in multibacillary leprosy patients. These stable, standardized plates can significantly contribute to efficient leprosy serology research and support its widespread distribution and use in endemic countries.

Screening and analysis for potential clinical diagnostic and prognostic markers in allergic rhinitis.

PURPOSE: To identify potential clinical diagnostic and prognostic markers for allergic rhinitis (AR) by analyzing a range of inflammatory and clinical markers in a cohort of patients. METHODS: We conducted a retrospective analysis of clinical data from 493 AR patients treated at Qianjiang Central Hospital from January to March 2023. Patients were categorized based on their outcome. Inclusion and exclusion criteria were strictly applied to select the study population. Various clinical and inflammatory markers were assessed, and statistical analyses were performed to evaluate their diagnostic and prognostic utility. RESULTS: No significant differences in traditional demographic factors were found between the good and poor prognosis groups (all P > 0.05). However, significant differences were observed in several inflammatory and clinical markers: Interleukin-4 (IL-4) levels were 17.32 ± 4.21 pg/mL in the good prognosis group versus 18.56 ± 5.89 pg/mL in the poor prognosis group (t=2.562, P=0.011). Interleukin-5 (IL-5) levels were 15.65 ± 3.78 pg/mL versus 16.52 ± 4.56 pg/mL, respectively (t=2.221, P=0.027). Transforming growth factor-ß1 (TGF-ß1) levels were 39.16 ± 8.92 pg/mL versus 41.32 ± 9.67 pg/mL (t=2.513, P=0.012), and histamine levels were 11.87 ± 3.21 ng/mL versus 12.56 ± 4.03 ng/mL (t=1.991, P=0.047). Interleukin-13 (IL-13) levels were 16.32 ± 3.56 pg/mL versus 17.09 ± 4.21 pg/mL (t=2.108, P=0.036). Serum immunoglobulin E (IgE) levels were significantly different, with 164.87 ± 45.32 IU/mL in the good prognosis group compared to 198.56 ± 58.21 IU/mL in the poor prognosis group (t=6.866, P < 0.001). The composite biomarker model demonstrated high predictive value for AR prognosis with an Area Under Curve of 0.906. Individual markers such as TGF-ß1, IL-13, and serum IgE levels showed strong diagnostic potential. CONCLUSION: Our findings underscore the clinical utility of various inflammatory and clinical markers as diagnostic and prognostic indicators for AR. TGF-ß1, IL-13, and serum IgE levels, in particular, demonstrated significant diagnostic and prognostic value. An integrated approach combining multiple biomarkers could enhance the accuracy of AR diagnosis and prognosis. Further validation through prospective clinical studies and consideration of treatment interventions are recommended to clarify the clinical implications of these markers.

Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles.

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

Determination of dysbiosis by the method of multiplex real-time polymerase chain reaction in chronic catarrhal gingivitis in children.

OBJECTIVE: Aim: The aim of the study was to determine the quantitative and qualitative characteristics of the microbiota of dento-gingival plaque in children to improve the quality of treatment of chronic catarrhal gingivitis. PATIENTS AND METHODS: Materials and Methods: It was examined 16 children aged 9-16 years with a diagnosis of K05.1: chronic gingivitis and 10 persons with intact gums were taken as a comparison group. A clinical dental examination was performed on the study participants and a sample was taken to determine the bacteria in the periodontal plaque. RESULTS: Results: The results of statistical processing of the research data allowed us to establish that in patients with chronic gingivitis, quantitative indicators of the total bacterial mass, Lactobacillus spp., Enterobacteriaceae, Gardnerella vaginalis/Prevotella bivia/Porphyromonas spp. in the sample of periodontal plaque significantly exceeded the indicators of healthy patients. It was determined that the examined children with chronic gingivitis, the total number of Lactobacillus spp. significantly exceeds its amount in people with intact gums. CONCLUSION: Conclusions: The changes in the quantitative and qualitative characteristics of the main representatives of the microf i lm of dento-gingival plaque, which characterize dysbiosis, are of signif i cant clinical signif i cance. Study of the quantitative characteristics of Lactobacterium spp., Enterobacterium spp., Streptococcacea spp., Gardnerella spp., Prevotella spp., Porphyromonas spp., Eubacteridacea spp., Mycoplasma (hominis + genitalium), Candida spp. is a diagnostic factor in determining the condition of the mucous membrane of the oral cavity.

Transmission of Brucella canis in a canine kennel following introduction of an infected dog.

Brucella canis is a zoonotic pathogen and the main causative agent of canine brucellosis. In the Netherlands, B. canis had previously only been detected in individual cases of imported dogs. However, an outbreak of B. canis occurred for the first time in a cohort of autochthonous dogs in a breeding kennel in 2019. The outbreak began with a positive serological test result of an imported intact male dog showing clinical symptoms of brucellosis. Consequently, urine and blood samples were collected and tested positive for B. canis by culture, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and whole-genome-sequencing (WGS). Screening of the contact dogs in the kennel where the index case was kept, revealed that antibodies against B. canis could be detected in 23 out of 69 dogs (34 %) by serum agglutination test (SAT). Of the 23 seropositive dogs, B. canis could be cultured from the urine and/or heparin samples of 19 dogs (83 %). This outbreak represents the first documented case of transmission of B. canis to autochthonous contact dogs in the Netherlands. WGS revealed all B. canis isolates belonged to the same cluster, which means the transmission of B. canis in the breeding kennel was most likely caused by the introduction of one infected dog. Comparing this cluster with data from other B. canis isolates, it also appears that characteristic clusters of B. canis are present in several endemic countries. These clusters seem to remain stable over time and may help in locating the origin of new isolates found. This outbreak showed that the international movement of dogs from endemic countries poses a threat to the canine population, while serological screening and WGS proved to be valuable tools for respectively screening and the epidemiological investigation.

Zoonotic pathogens in wild Asian primates: a systematic review highlighting research gaps.

Introduction: Ongoing global changes, including natural land conversion for agriculture and urbanization, modify the dynamics of human-primate contacts, resulting in increased zoonotic risks. Although Asia shelters high primate diversity and experiences rapid expansion of human-primate contact zones, there remains little documentation regarding zoonotic surveillance in the primates of this region. Methods: Using the PRISMA guidelines, we conducted a systematic review to compile an inventory of zoonotic pathogens detected in wild Asian primates, while highlighting the coverage of primate species, countries, and pathogen groups surveyed, as well as the diagnostic methods used across the studies. Moreover, we compared the species richness of pathogens harbored by primates across diverse types of habitats classified according to their degree of anthropization (i.e., urban vs. rural vs. forest habitats). Results and discussion: Searches of Scopus, PubMed, and the Global Mammal Parasite Database yielded 152 articles on 39 primate species. We inventoried 183 pathogens, including 63 helminthic gastrointestinal parasites, two blood-borne parasites, 42 protozoa, 45 viruses, 30 bacteria, and one fungus. Considering each study as a sample, species accumulation curves revealed no significant differences in specific richness between habitat types for any of the pathogen groups analyzed. This is likely due to the insufficient sampling effort (i.e., a limited number of studies), which prevents drawing conclusive findings. This systematic review identified several publication biases, particularly the uneven representation of host species and pathogen groups studied, as well as a lack of use of generic diagnostic methods. Addressing these gaps necessitates a multidisciplinary strategy framed in a One Health approach, which may facilitate a broader inventory of pathogens and ultimately limit the risk of cross-species transmission at the human-primate interface. Strengthening the zoonotic surveillance in primates of this region could be realized notably through the application of more comprehensive diagnostic techniques such as broad-spectrum analyses without a priori selection.

KCL TEST: an open-source inspired asymptomatic SARS-CoV-2 surveillance programme in an academic institution.

Rapid and accessible testing was paramount in the management of the COVID-19 pandemic. Our university established KCL TEST: a SARS-CoV-2 asymptomatic testing programme that enabled sensitive and accessible PCR testing of SARS-CoV-2 RNA in saliva. Here, we describe our learnings and provide our blueprint for launching diagnostic laboratories, particularly in low-resource settings. Between December 2020 and July 2022, we performed 158277 PCRs for our staff, students, and their household contacts, free of charge. Our average turnaround time was 16 h and 37 min from user registration to result delivery. KCL TEST combined open-source automation and in-house non-commercial reagents, which allows for rapid implementation and repurposing. Importantly, our data parallel those of the UK Office for National Statistics, though we detected a lower positive rate and virtually no delta wave. Our observations strongly support regular asymptomatic community testing as an important measure for decreasing outbreaks and providing safe working spaces. Universities can therefore provide agile, resilient, and accurate testing that reflects the infection rate and trend of the general population. Our findings call for the early integration of academic institutions in pandemic preparedness, with capabilities to rapidly deploy highly skilled staff, as well as develop, test, and accommodate efficient low-cost pipelines.

Rhinomanometry: A Comprehensive Review of Its Applications and Advancements in Rhinology Practice.

Rhinomanometry is a pivotal diagnostic technique in rhinology, providing a quantitative assessment of nasal airflow and resistance. This review comprehensively examines the historical development, principles and clinical applications of rhinomanometry, emphasising its role in diagnosing nasal obstructions, preoperative evaluations and monitoring therapeutic outcomes. Recent advancements, including the integration with imaging technologies and the application of artificial intelligence (AI), have significantly enhanced the accuracy and utility of rhinomanometry. Despite facing challenges such as technical limitations and the need for standardisation, rhinomanometry remains an invaluable tool in both clinical and research settings. The review also explores future directions, highlighting the potential for device miniaturisation, telemedicine integration, personalised protocols and collaborative research efforts. These advancements will likely expand the accessibility, accuracy and clinical relevance of rhinomanometry, solidifying its importance in the ongoing evolution of rhinology practice.

Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.

Neurological phenotypes and treatment outcomes in Eagle syndrome: systematic review and meta-analysis.

Background: Eagle syndrome is caused by an elongated styloid process affecting carotid arteries and cranial nerves. Pain, dysphagia, tinnitus, paresthesia (classic subtype), and neurovascular events (vascular subtype) may be triggered by head movements or arise spontaneously. However, Eagle syndrome remains underappreciated in the neurological community. We aimed to determine the most common neurological and non-neurological clinical presentations in patients with Eagle syndrome and to assess the clinical outcome post-surgical resection in comparison to non-surgical therapies. Methodology: We conducted a systematic review of patient-level data on adults with Eagle syndrome, following PRISMA guidelines. We extracted data on demographics, presenting symptoms, neurological deficits, radiological findings, and treatments, including outcomes and complications, from studies in multiple indexing databases published between 2000 and 2023. The study protocol is registered with PROSPERO. Results: In total, 285 studies met inclusion criteria, including 497 patients with Eagle syndrome (mean age 47.3 years; 49.8% female). Classical Eagle (370 patients, 74.5%) was more frequent than vascular Eagle syndrome (117 patients, 23.5%, p < 0.0001). Six patients (1.2%) presented with both variants and the subvariant for four patients (0.8%) was unknown. There was a male preponderance (70.1% male) in the vascular subtype. A history of tonsillectomy was more frequent in classic (48/153 cases) than in vascular (2/33 cases) Eagle syndrome (Odds Ratio 5.2, 95% CI [1.2-22.4]; p = 0.028). By contrast, cervical movements as trigger factors were more prevalent in vascular (12/33 cases) than in classic (7/153 cases) Eagle syndrome (Odds Ratio 7.95, 95% CI [2.9-21.7]; p = 0.0001). Headache and Horner syndrome were more frequent in vascular Eagle syndrome and dysphagia and neck pain more prominent in classic Eagle syndrome (all p < 0.01). Surgically treated patients achieved overall better outcomes than medically treated ones: Eighty-one (65.9%) of 123 medically treated patients experienced improvement or complete resolution, while the same applied to 313 (97.8%) of 320 surgical patients (Odds Ratio 1.49, 95% CI [1.1-2.0]; p = 0.016). Conclusions: Eagle syndrome is underdiagnosed with potentially serious neurovascular complications, including ischemic stroke. Surgical treatment achieves better outcomes than conservative management. Although traditionally the domain of otorhinolaryngologist, neurologist should include this syndrome in differential diagnostic considerations because of the varied neurological presentations that are amenable to effective treatment.

Rapid Drug Susceptibility Testing to Preserve Antibiotics.

Antibiotic resistance is a global challenge likely to cost trillions of dollars in excess costs in the health system and more importantly, millions of lives every year. A major driver of resistance is the absence of susceptibility testing at the time a healthcare worker needs to prescribe an antimicrobial. The effect is that many prescriptions are unintentionally wasted and expose mutable organisms to antibiotics increasing the risk of resistance emerging. Often simplistic solutions are applied to this growing issue, such as a naïve drive to increase the speed of drug susceptibility testing. This puts a spotlight on a technological solution and there is a multiplicity of such candidate DST tests in development. Yet, if we do not define the necessary information and the speed at which it needs to be available in the clinical decision-making progress as well as the necessary integration into clinical pathways, then little progress will be made. In this chapter, we place the technological challenge in a clinical and systems context. Further, we will review the landscape of some promising technologies that are emerging and attempt to place them in the clinic where they will have to succeed.

An exhaled breath gas sensor system for near-infrared ammonia measurement and kidney diagnostics.

Breath analysis enables rapid, noninvasive diagnosis of human health by identifying and quantifying exhaled biomarker. Here, we demonstrated an exhaled breath sensing method using the near-infrared laser spectroscopy, and sub parts-per-million (ppm) level ammonia detection inside the exhaled gas was achieved employing a distributed feedback laser centered at 1512 nm and Kalman filtering algorithm. Integration of the ammonia sensor was realized for exhaled breath analysis of kidney patients, and a dual operation mechanism with static and dynamic modes was proposed to make this method applicable for real-time and comprehensive pre-diagnosis of kidney disease.

Fusarium and Neocosmospora: fungal priority pathogens in laboratory diagnosis.

Fusarium and Neocosmospora are two fungal genera recently recognized in the list of fungal priority pathogens. They cause a wide range of diseases that affect humans, animals, and plants. In clinical laboratories, there is increasing concern about diagnosis due to limitations in sample collection and morphological identification. Despite the advances in molecular diagnosis, due to the cost, some countries cannot implement these methodologies. However, recent changes in taxonomy and intrinsic resistance to antifungals reveal the necessity of accurate species-level identification. In this review, we discuss the current phenotypic and molecular tools available for diagnosis in clinical laboratory settings and their advantages and disadvantages.

Specificity of the short-story task for autism diagnosis when controlling for depression.

Securing an accurate autism-spectrum-condition diagnosis, particularly among women, remains challenging for autistic adults. Building upon previous research highlighting the short-story task (SST) as a promising tool for detecting fiction-based mentalizing difficulties in autistic adults, this study expands its scope. We investigated the SST's discriminative capacity across three distinct groups: autistic individuals (n = 32), nonautistic individuals without mental health problems (n = 32), and nonautistic individuals with clinical depression (n = 30). All three groups differed significantly from each other in their SST mentalizing score with the nonautistic group having the highest scores, the nonautistic but depressed group having medium scores and the autistic group showing the lowest scores. Receiver operator curve (ROC) analysis reaffirmed the SST's efficacy as a discriminator. Moreover, a linear regression analysis identified the SST mentalizing score, the SST comprehension score, and the number of books read per month as significant predictors of autism-spectrum-condition diagnosis. These findings bolster the SST's potential as a valuable adjunct in autism diagnostics, highlighting its discriminatory ability across diverse samples.

Improved Diagnosis and Treatment Monitoring of Tuberculosis Using Stool and the Tuberculosis Bacterial Load Assay (TB-MBLA).

The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool.

Review of Pseudoloma neurophilia (Microsporidia): A common neural parasite of laboratory zebrafish (Danio rerio).

Zebrafish (Danio rerio) is now the second most used animal model in biomedical research. As with other vertebrate models, underlying diseases and infections often impact research. Beyond mortality and morbidity, these conditions can compromise research end points by producing nonprotocol induced variation within experiments. Pseudoloma neurophilia, a microsporidium that targets the central nervous system, is the most frequently diagnosed pathogen in zebrafish facilities. The parasite undergoes direct, horizontal transmission within populations, and is also maternally transmitted with spores in ovarian fluid and occasionally within eggs. This transmission explains the wide distribution among research laboratories as new lines are generally introduced as embryos. The infection is chronic, and fish apparently never recover following the initial infection. However, most fish do not exhibit outward clinical signs. Histologically, the parasite occurs as aggregates of spores throughout the midbrain and spinal cord and extends to nerve roots. It often elicits meninxitis, myositis, and myodegeneration when it infects the muscle. There are currently no described therapies for the parasite, thus the infection is best avoided by screening with PCR-based tests and removal of infected fish from a facility. Examples of research impacts include reduced fecundity, behavioral changes, transcriptome alterations, and autofluorescent lesions.

Revolutionary Point-of-Care Wearable Diagnostics for Early Disease Detection and Biomarker Discovery through Intelligent Technologies.

Early-stage disease detection, particularly in Point-Of-Care (POC) wearable formats, assumes pivotal role in advancing healthcare services and precision-medicine. Public benefits of early detection extend beyond cost-effectively promoting healthcare outcomes, to also include reducing the risk of comorbid diseases. Technological advancements enabling POC biomarker recognition empower discovery of new markers for various health conditions. Integration of POC wearables for biomarker detection with intelligent frameworks represents ground-breaking innovations enabling automation of operations, conducting advanced large-scale data analysis, generating predictive models, and facilitating remote and guided clinical decision-making. These advancements substantially alleviate socioeconomic burdens, creating a paradigm shift in diagnostics, and revolutionizing medical assessments and technology development. This review explores critical topics and recent progress in development of 1) POC systems and wearable solutions for early disease detection and physiological monitoring, as well as 2) discussing current trends in adoption of smart technologies within clinical settings and in developing biological assays, and ultimately 3) exploring utilities of POC systems and smart platforms for biomarker discovery. Additionally, the review explores technology translation from research labs to broader applications. It also addresses associated risks, biases, and challenges of widespread Artificial Intelligence (AI) integration in diagnostics systems, while systematically outlining potential prospects, current challenges, and opportunities.

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA.

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'→5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Streamlined two-step fragment analysis PCR and exome sequencing of RFC1 for diagnostic testing of suspected CANVAS patients.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two-step PCR consisting of a short-allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non-CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two-step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow.