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A molecular diagnosis from the analysis of sequencing data in rare Mendelian diseases has a huge impact on the management of patients and their families. Numerous patient phenotype-aware variant prioritisation (VP) tools have been developed to help automate this process, and shorten the diagnostic odyssey, but performance statistics on real patient data are limited. Here we identify, assess, and compare the performance of all up-to-date, freely available, and programmatically accessible tools using a whole-exome, retinal disease dataset from 134 individuals with a molecular diagnosis. All tools were able to identify around two-thirds of the genetic diagnoses as the top-ranked candidate, with LIRICAL performing best overall. Finally, we discuss the challenges to overcome most cases remaining undiagnosed after current, state-of-the-art practices.
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Exoma , Enfermedades Raras , Humanos , Fenotipo , Secuenciación del Exoma , Enfermedades Raras/diagnóstico , Enfermedades Raras/genéticaRESUMEN
To understand the genetic contribution to primary pediatric cardiomyopathy, we performed exome sequencing in a large cohort of 528 children with cardiomyopathy. Using clinical interpretation guidelines and targeting genes implicated in cardiomyopathy, we identified a genetic cause in 32% of affected individuals. Cardiomyopathy sub-phenotypes differed by ancestry, age at diagnosis, and family history. Infants < 1 year were less likely to have a molecular diagnosis (p < 0.001). Using a discovery set of 1,703 candidate genes and informatic tools, we identified rare and damaging variants in 56% of affected individuals. We see an excess burden of damaging variants in affected individuals as compared to two independent control sets, 1000 Genomes Project (p < 0.001) and SPARK parental controls (p < 1 × 10-16). Cardiomyopathy variant burden remained enriched when stratified by ancestry, variant type, and sub-phenotype, emphasizing the importance of understanding the contribution of these factors to genetic architecture. Enrichment in this discovery candidate gene set suggests multigenic mechanisms underlie sub-phenotype-specific causes and presentations of cardiomyopathy. These results identify important information about the genetic architecture of pediatric cardiomyopathy and support recommendations for clinical genetic testing in children while illustrating differences in genetic architecture by age, ancestry, and sub-phenotype and providing rationale for larger studies to investigate multigenic contributions.
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Cardiomiopatía Dilatada/genética , Exoma , Regulación de la Expresión Génica , Genotipo , Patrón de Herencia , Edad de Inicio , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Estudios de Casos y Controles , Niño , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Variación Genética , Humanos , Masculino , Fenotipo , Guías de Práctica Clínica como Asunto , Secuenciación del ExomaRESUMEN
BACKGROUND: Malarial infections are often missed by microscopy, and most parasite carriers are asymptomatic in low-endemicity settings. Whether parasite detectability and its ability to elicit symptoms change as transmission declines remains unclear. METHODS: We performed a prospective panel survey with repeated measurements on the same participants over 12 months to investigate whether Plasmodium vivax detectability by microscopy and risk of symptoms upon infection varied during a community-wide larviciding intervention in the Amazon basin of Brazil that markedly reduced vector density. We screened 1096 to 1400 residents in the intervention site for malaria by microscopy and quantitative TaqMan assays at baseline and twice during intervention. RESULTS: We found that more P vivax infections than expected from their parasite densities measured by TaqMan assays were missed by microscopy as transmission decreased. At lower transmission, study participants appeared to tolerate higher P vivax loads without developing symptoms. We hypothesize that changes in the ratio between circulating parasites and those that accumulate in the bone marrow and spleen, by avoiding peripheral blood microscopy detection, account for decreased parasite detectability and lower risk of symptoms under low transmission. CONCLUSIONS: P vivax infections are more likely to be subpatent and remain asymptomatic as malaria transmission decreases.
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Malaria Falciparum , Malaria Vivax , Malaria , Humanos , Malaria Vivax/parasitología , Brasil/epidemiología , Estudios Prospectivos , Malaria Falciparum/parasitología , Prevalencia , Plasmodium vivax , Plasmodium falciparumRESUMEN
Urine-based testing is promising for noninvasive diagnosis of urothelial carcinoma (UC) but has suboptimal sensitivity for early-stage tumors. Herein, we developed a multitarget urine tumor DNA test, UI-Seek, for UC detection and evaluated its clinical feasibility. The prediction model was developed in a retrospective cohort (n = 382), integrating assays for FGFR3 and TERT mutations and aberrant ONECUT2 and VIM methylation to generate a UC-score. The test performance was validated in a double-blinded, multicenter, prospective trial (n = 947; ChiCTR2300076543) and demonstrated a sensitivity of 91.37% and a specificity of 95.09%. The sensitivity reached 75.81% for low-grade Ta tumors and exceeded 93% in high-grade Ta and higher stages (T1 to T4). Simultaneous identification of both bladder and upper urinary tract tumors was enabled with sensitivities exceeding 90%. No significant confounding effects were observed regarding benign urological diseases or non-UC malignancies. The test showed improved sensitivities over urine cytology, the NMP22 test, and UroVysion FISH alongside comparable specificities. The single-target accuracy was greater than 98% as confirmed by Sanger sequencing. Post-surgery UC-score decreased in 97.7% of subjects. Overall, UI-Seek demonstrated robust performance and considerable potential for the early detection of UC.
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Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Carcinoma de Células Transicionales/diagnóstico , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/orina , Estudios Retrospectivos , Estudios Prospectivos , Sensibilidad y Especificidad , Resultado del Tratamiento , ADN , Biomarcadores de Tumor/genética , Factores de Transcripción , Proteínas de HomeodominioRESUMEN
Candida auris was reported by the WHO as second to Cryptococcus neoformans, in the list of nineteen fungal priority pathogens, along with two species with a new nomenclature, Nakaseomyces glabrata (Candida glabrata) and Pichia kudriavzevii (Candida krusei). This novel classification was based on antifungal resistance, the number of deaths, evidence-based treatment, access to diagnostics, annual incidence, and complications and sequelae. We assessed which molecular assays have been used to diagnose Candida auris outbreaks in the last five years. Using "Candida auris; outbreak; molecular detection" as keywords, our search in PubMed revealed 32 results, from which we selected 23 original papers published in 2019-2024. The analyzed studies revealed that the detection methods were very different: from the VITEK® 2 System to MALDI TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight), NGS (Next-Generation Sequencing), WGS (Whole Genome Sequencing), and commercially available real-time PCR (Polymerase Chain Reaction) assays. Moreover, we identified studies that detected antifungal resistance genes (e.g., FKS for echinocandins and ERG11 for azoles). The analyzed outbreaks were from all continents, which confirms the capability of this yeast to spread between humans and to contaminate the environment. It is important that real-time PCR assays were developed for accurate and affordable detection by all laboratories, including the detection of antifungal resistance genes. This will allow the fast and efficient implementation of stewardship programs in hospitals.
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Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic defects in cortisol synthesis and shows elevated ACTH concentrations, which in turn has downstream effects. The most common variant of CAH, 21-hydroxylase deficiency (21OHD), is the result of pathogenic variants in the CYP21A2 gene and is one of the most common monogenic disorders. However, the genetics of 21OHD is complex and challenging. The CYP21A2 gene is located in the RCCX copy number variation (CNV), a complex, multiallelic, and tandem CNV in the major histocompatibility complex (MHC) class III region on chromosome 6 (band 6p21.3). Here, CYP21A2 and its pseudogene CYP21A1P are located 30 kb apart and share a high nucleotide homology of approximately 98% and 96% in exons and introns, respectively. This high-sequence homology facilitates large structural rearrangements, copy number changes, and gene conversion through intergenic recombination. There is a good genotype-phenotype correlation in 21OHD, and genotyping can be performed to confirm the clinical diagnosis, predict long-term outcomes, and determine genetic counseling. Thus, genotyping in CAH is clinically relevant but the interpretations can be challenging for non-initiated clinicians. Here, there are some concrete examples of how molecular diagnosis can sometimes require the use of multiple molecular strategies.
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Many inherited conditions cause hepatocellular cholestasis in infancy, including progressive familial intrahepatic cholestasis (PFIC), a heterogeneous group of diseases with highly overlapping symptoms. In our study, six unrelated Tunisian infants with PFIC suspicion were the subject of a panel-target sequencing followed by an exhaustive bioinformatic and modeling investigations. Results revealed five disease-causative variants including known ones: (the p.Asp482Gly and p.Tyr354 * in the ABCB11 gene and the p.Arg446 * in the ABCC2 gene), a novel p.Ala98Cys variant in the ATP-binding cassette subfamily G member 5 (ABCG5) gene and a first homozygous description of the p.Gln312His in the ABCB11 gene. The p.Gln312His disrupts the interaction pattern of the bile salt export pump as well as the flexibility of the second intracellular loop domain harboring this residue. As for the p.Ala98Cys, it modulates both the interactions within the first nucleotide-binding domain of the bile transporter and its accessibility. Two additional potentially modifier variants in cholestasis-associated genes were retained based on their pathogenicity (p.Gly758Val in the ABCC2 gene) and functionality (p.Asp19His in the ABCG8 gene). Molecular findings allowed a PFIC2 diagnosis in five patients and an unexpected diagnosis of sisterolemia in one case. The absence of genotype/phenotype correlation suggests the implication of environmental and epigenetic factors as well as modifier variants involved directly or indirectly in the bile composition, which could explain the cholestasis phenotypic variability.
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Colestasis Intrahepática , Colestasis , Lactante , Humanos , Recién Nacido , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Colestasis Intrahepática/diagnóstico , Colestasis Intrahepática/genética , Colestasis/genética , Estudios de Asociación Genética , Mutación , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Lipoproteínas/genéticaRESUMEN
The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.
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Toxoplasma , Toxoplasmosis Congénita , Toxoplasmosis , Humanos , Toxoplasma/genética , Toxoplasmosis/diagnóstico , Toxoplasmosis/parasitología , Toxoplasmosis Congénita/diagnóstico , Toxoplasmosis Congénita/parasitología , ADN , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , ADN Protozoario/genética , ADN Protozoario/análisisRESUMEN
Early initiation of antimicrobial therapy targeting resistant bacterial pathogens causing sepsis and bloodstream infections (BSIs) is critical for a successful outcome. The T2Resistance Panel (T2R) detects the following resistance genes within organisms that commonly cause BSIs directly from patient blood samples: blaKPC, blaCTXM-14/15, blaNDM/bla/IMP/blaVIM, blaAmpC, blaOXA, vanA, vanB, and mecA/mecC. We conducted a prospective study in two major medical centers for the detection of circulating resistance genes by T2R in patients with BSIs. T2R reports were compared to antimicrobial susceptibility testing (AST), phenotypic identification, and standard molecular detection assays. Among 59 enrolled patients, 25 resistance genes were identified: blaKPC (n = 10), blaNDM/bla/IMP/blaVIM (n = 5), blaCTXM-14/15 (n = 4), blaAmpC (n = 2), and mecA/mecC (n = 4). Median time-to-positive-T2R in both hospitals was 4.4 hours [interquartile range (IQR): 3.65-4.97 hours] in comparison to that for positive blood cultures with final reporting of AST of 58.34 h (IQR: 45.51-111.2 hours; P < 0.0001). The sensitivity of T2R to detect the following genes in comparison to AST was 100% for blaCTXM-14/15, blaNDM/bla/IMP/blaVIM, blaAmpC, mecA/mecC and 87.5% for blaKPC. When monitored for the impact of significant antimicrobial changes, there were 32 events of discontinuation of unnecessary antibiotics and 17 events of escalation of antibiotics, including initiation of ceftazidime/avibactam in six patients in response to positive T2R results for blaKPC. In summary, T2R markers were highly sensitive for the detection of drug resistance genes in patients with bacterial BSIs, when compared with standard molecular resistance detection systems and phenotypic identification assays while significantly reducing by approximately 90% the time to detection of resistance compared to standard methodology and impacting clinical decisions for antimicrobial therapy. IMPORTANCE: This is the first reported study to our knowledge to identify key bacterial resistance genes directly from the bloodstream within 3 to 5 hours in patients with bloodstream infections and sepsis. The study further demonstrated a direct effect in modifying initial empirical antibacterial therapy in response to T2R signal to treat resistant bacteria causing bloodstream infections and sepsis.
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Antiinfecciosos , Bacteriemia , Infecciones Bacterianas , Sepsis , Humanos , Estudios Prospectivos , Bacteriemia/microbiología , Proyectos Piloto , Bacterias/genética , Antibacterianos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: Studying the sensitisation profiles of patients with allergies allows for a deeper understanding of the disease which may facilitate the selection of the best-personalised allergen immunotherapy. This observational, cross-sectional, multicentre study aimed to demonstrate the heterogeneity of the German population with allergies by analysing specific immunoglobulin E (sIgE) patterns towards aeroallergens and exploring the relationship between sensitisation and clinical symptoms. METHODS: In total, 500 patients with allergies from different regions of Germany were recruited based on their case histories, clinical allergic symptoms and skin prick test data for aeroallergens. Serum samples were analysed using ImmunoCAP assays to determine sIgE levels for 33 allergenic sources and 43 molecular allergens. RESULTS: Most patients (81%) were polysensitised. Betula verrucosa pollen was the most common cause of sensitisation (59%), followed by Phleum pratense (58%) and Dermatophagoides pteronyssinus (44%). The highest prevalence rates of molecular allergens were observed for Bet v 1 (84%) from birch pollen, Phl p 1 from grass pollen (82%), Der p 2 (69%) from mites and Fel d 1 (69%) from cat. Polysensitisation was significantly associated with the presence of asthma and the severity of rhinitis symptoms. CONCLUSIONS: Our findings show a high rate of polysensitisation and emphasise the importance of molecular diagnosis for more precise and comprehensive insights into sensitisation patterns and their association with clinical symptoms. These data may help improve personalised diagnosis and immunotherapy adapted to the needs of individual patients in the region.
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Alérgenos , Hipersensibilidad , Inmunoglobulina E , Humanos , Masculino , Femenino , Adulto , Inmunoglobulina E/inmunología , Inmunoglobulina E/sangre , Persona de Mediana Edad , Estudios Transversales , Alérgenos/inmunología , Hipersensibilidad/inmunología , Hipersensibilidad/epidemiología , Hipersensibilidad/diagnóstico , Hipersensibilidad/terapia , Alemania/epidemiología , Adolescente , Adulto Joven , Anciano , Pruebas Cutáneas , Índice de Severidad de la Enfermedad , Inmunización , Polen/inmunología , PrevalenciaRESUMEN
Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 µ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.
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Carcinoma de Pulmón de Células no Pequeñas , Receptores ErbB , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Pulmonares , Mutación , Humanos , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microfluídica/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Técnicas Analíticas Microfluídicas/métodos , Adhesión en ParafinaRESUMEN
The molecular composition of exhaled human breath can reflect various physiological and pathological conditions. Considerable progress has been achieved over the past decade in real-time analysis of exhaled human breath using direct mass spectrometry methods, including selected ion flow tube mass spectrometry, proton transfer reaction mass spectrometry, extractive electrospray ionization mass spectrometry, secondary electrospray ionization mass spectrometry, acetone-assisted negative photoionization mass spectrometry, atmospheric pressure photoionization mass spectrometry, and low-pressure photoionization mass spectrometry. Here, recent developments in direct mass spectrometry analysis of exhaled human breath are reviewed with regard to analytical performance (chemical sensitivity, selectivity, quantitative capabilities) and applications of the developed methods in disease diagnosis, targeted molecular detection, and real-time metabolic monitoring.
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Covid-19 in West Africa masked outbreaks of vaccine-preventable diseases such as the measles epidemic in children in Guinea in 2021-2022 characterized by a lack of confirmation of suspected clinical cases. During weeks 13-22 of 2022, saliva samples were collected from 213 children (3-60 months old) with measles-like symptoms within the St Gabriel dispensary in Conakry. Samples were processed in Virus Transport Medium (VTM) and tested on the same day by triplex reverse transcriptase -real-time polymerase chain reaction for Measles, Rubella and RNaseP. Samples were also tested for HHV6 and Parvovirus B19, viruses causing clinical signs similar to measles. We confirmed 146 (68.5%) measles cases, 27 (12.7%) rubella, 5 (2.3%) double-positive measles-rubella, 35 (16.4%) HHV-6 and 8 (3.75%) Parvovirus B19. To test the assay's robustness, 27 samples were kept at 26-30°C. Measles and rubella were still detected after 7 days at 26-30°C, and after 21 days measles and rubella were still detectable in all samples but one. Sequencing indicated the circulation of the B3 measles genotype, as expected in West Africa. This study highlights the robustness of the measles/rubella diagnostic test on saliva samples stored in VTM. The high level of rubella detection questioned the single valence measles vaccination strategy.
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COVID-19 , Exantema , Herpesvirus Humano 6 , Sarampión , Parvovirus B19 Humano , Rubéola (Sarampión Alemán) , Niño , Humanos , Lactante , Preescolar , Papúa Nueva Guinea , Anticuerpos Antivirales , Inmunoglobulina M , COVID-19/epidemiología , COVID-19/complicaciones , Guinea , Virus del Sarampión/genética , Parvovirus B19 Humano/genéticaRESUMEN
Copy number variants (CNVs) remain a major etiological cause of neurodevelopmental delay and congenital malformations. Chromosomal microarray analysis (CMA) represents the gold standard for CNVs molecular characterization. We applied CMA throughout the patient's clinical diagnostic workup, as the patient's medical provider requested. We collected CMA results of 3380 patients enrolled for 5 years (2016-2021). We found 830 CNVs in 719 patients with potential clinical significance, that is, (i) pathogenic, (ii) likely pathogenic, and (iii) variants of uncertain significance (VUS), from which 10.6% (predominantly involving chromosomes 15 and 22) were most likely the final cause underpinning the patients' clinical phenotype. For those associated with neurodevelopmental phenotypes, the rate of pathogenic or likely pathogenic findings among the patients with CNVs was 60.75%. When considering epileptic phenotypes, it was 59%. Interestingly, our protocol identified two gains harbored in 17q21.31 and 9q34.3, internationally classified initially as VUS. However, because of their high frequency, we propose that these two VUS be reclassified as likely benign in this widely heterogeneous phenotypic population. These results support the diagnostic yield efficiency of CMA in characterizing CNVs to define the final molecular cause of genetic diseases in this cohort of Colombian patients, the most significant sample of patients from a Latino population, and define new benign polymorphic CNVs.
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Aberraciones Cromosómicas , Cromosomas , Humanos , Análisis por Micromatrices , Cromosomas Humanos Par 15 , Variaciones en el Número de Copia de ADN/genéticaRESUMEN
TANGO2 deficiency disorder (TDD) is a rare, autosomal recessive condition caused by pathogenic variants in TANGO2, a gene residing within the region commonly deleted in 22q11.2 deletion syndrome (22q11.2DS). Although patients with 22q11.2DS are at substantially higher risk for comorbid TDD, it remains underdiagnosed within 22q11.2DS, likely due to overlapping symptomatology and a lack of knowledge about TDD. Initiation of B-vitamin supplementation may provide therapeutic benefit in TDD, highlighting the need for effective screening methods to improve diagnosis rates in this at-risk group. In this retrospective, multicenter study, we evaluated two cohorts of patients with 22q11.2DS (total N = 435) for possible comorbid TDD using two different symptom-based screening methods (free text-mining and manual chart review versus manual chart review alone). The methodology of the cohort 1 screening method successfully identified a known 22q11.2DS patient with TDD. Combined, these two cohorts identified 21 living patients meeting the consensus recommendation for TANGO2 testing for suspected comorbid TDD. Of the nine patients undergoing TANGO2 sequencing with del/dup analysis, none were ultimately diagnosed with TDD. Of the 12 deaths in the suspected comorbid TDD cohort, some of these patients exhibited symptoms (rhabdomyolysis, cardiac arrhythmia, or metabolic crisis) suspicious of comorbid TDD contributing to their death. Collectively, these findings highlight the need for robust prospective screening tools for diagnosing comorbid TDD in patients with 22q11.2DS.
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Trichophyton indotineae is an emerging species of the Trichophyton mentagrophytes complex (TMC), responsible for an epidemic of widespread hairless skin infections that is frequently (50-70%) resistant to terbinafine. In order to initiate appropriate treatment as quickly as possible without waiting for culture positivity (10-15 days) and molecular identification from the strain, we developed a dual quantitative PCR (qPCR) for the direct detection of T. indotineae in clinical samples. We first designed a T. indotineae-specific qPCR assay (TI-qPCR) targeting a single specific polymorphism in the internal transcribed spacer region. Although none of the 94 non-dermatophyte and 7 dermatophyte species were amplified, this TI-qPCR allowed amplification of other TMC species at a lower yield. With equal amounts (0.1 ng) of DNA per reaction, the mean quantitative cycle (Cq) values for T. indotineae and non-indotineae TMC were 27.9 (±0.1) and 38.9 (±0.3), respectively. Therefore, we normalized this assay against a previously validated pan-dermatophyte qPCR assay (PD-qPCR) and relied on the ΔCq [(TI-qPCR) - (PD-qPCR)] to identify T. indotineae versus other TMC species. Dual assay was validated using 86 clinical samples of culture-confirmed T. indotinea and 19 non-indotineae TMC cases. The mean ΔCq for non-indotineae TMC was 9.6 ± 2.7, whereas the ΔCq for T. indotinea was -1.46 ± 2.1 (P < .001). Setting the ΔCq at 4.5 as a cutoff value resulted in 100% specificity for the detection of T. indotineae. This dual qPCR assay quickly detects T. indotineae from skin scrapings, aiding in early diagnosis and treatment for patients with suspected infection.
Identifying the emerging species Trichophyton indotineae is long and requires to wait for culture positivity. We developed a dual qPCR strategy to detect T. indotineae directly from clinical sample with a 100% sensitivity.
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Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Tiña , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tiña/diagnóstico , Tiña/microbiología , ADN de Hongos/genética , Trichophyton/genética , Trichophyton/aislamiento & purificación , Trichophyton/clasificación , Técnicas de Diagnóstico Molecular/métodos , ADN Espaciador Ribosómico/genéticaRESUMEN
Zoonotic sporotrichosis caused by Sporothrix brasiliensis is an emerging mycosis in Latin America. One of the problems to quickly treat infected animals and break the transmission chain is associated with the time-consuming gold-standard diagnosis method (culture). We aimed to evaluate a species-specific polymerase chain reaction (PCR) for the diagnosis of sporotrichosis caused by S. brasiliensis using non-invasive samples. We performed a retrospective cross-sectional study using samples collected with swabs from humans and cats with clinical suspicion of sporotrichosis. Deoxyribonucleic acid (DNA) was extracted using a commercial kit, and a species-specific PCR for S. brasiliensis detection was performed. One hundred ten samples were included. PCR showed a good concordance with culture (86% of agreement) for human and cat samples (Kappa coefficient = 0.722, and 0.727, respectively). In conclusion, our data shows that this adapted PCR using non-invasive samples can be applied to sporotrichosis diagnosis, being a good alternative mainly in regions with a lack of mycologists to identify the fungus in culture, contributing to the control of this emergent zoonosis.
We aimed to evaluate a molecular method for diagnosing sporotrichosis caused by Sporothrix brasiliensis in humans and cats. We observed that the technique is in good agreement with the classic method and is a good alternative for assisting in the diagnosis and consequent control of this zoonosis.
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Enfermedades de los Gatos , Reacción en Cadena de la Polimerasa , Sporothrix , Esporotricosis , Esporotricosis/diagnóstico , Esporotricosis/microbiología , Esporotricosis/veterinaria , Gatos , Sporothrix/genética , Sporothrix/aislamiento & purificación , Sporothrix/clasificación , Humanos , Animales , Reacción en Cadena de la Polimerasa/métodos , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/microbiología , Estudios Retrospectivos , Estudios Transversales , Zoonosis/diagnóstico , Zoonosis/microbiología , ADN de Hongos/genética , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y EspecificidadRESUMEN
This was a cross-sectional study on the availability of laboratory infrastructure and capacity for the diagnosis of invasive fungal diseases in 24 public hospitals in Vietnam in 2023. Among the hospitals surveyed, 66.7% (14/21) had specialized personnel assigned for mycology testing, and 95.8% (23/24) had a separate microbiology laboratory space. Microscopy and culture methods are available in nearly all laboratories for isolate identification. Antifungal susceptibility testing is only performed for yeasts in 16/24 (66.7%) laboratories. Non-culture methods are hardly used in laboratories. Strengthening local laboratory capacities is essential to meeting health needs in these endemic regions.
There was a need for investment in fungal diagnostics to improve health services in the settings with a burden of endemic fungal infections.
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Hospitales Públicos , Infecciones Fúngicas Invasoras , Vietnam , Humanos , Estudios Transversales , Infecciones Fúngicas Invasoras/diagnóstico , Infecciones Fúngicas Invasoras/microbiología , Micología/métodos , Hongos/aislamiento & purificación , Hongos/clasificación , Hongos/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
In recent years, molecular biology-based diagnostic techniques have made remarkable strides and are now extensively utilized in clinical practice, providing invaluable insights for disease diagnosis and treatment. However, forensic medicine, especially forensic pathology, has witnessed relatively limited progress in the application of molecular biology technologies. A significant challenge in employing molecular techniques for forensic diagnoses lies in the quantitative and qualitative changes observed in diagnostic markers due to sample degradation-a recognized and formidable obstacle. Inspired by the success of DNA sequencing in forensic practices, which enables accurate individual identification even in cases involving degraded and deteriorated tissues and organs, we propose the application of the assay for transposase-accessible chromatin with sequencing (ATAC-seq) to identify targets at the transcriptional onset, exploring chromatin and DNA-level alterations for injury and disease inference in forensic samples. This study employs ATAC-seq to explore alterations in chromatin accessibility post-injury and their subsequent changes over a 2-h degradation period, employing traumatic brain injury (TBI) as a representative model. Our findings reveal high sensitivity of chromatin accessibility sites to injury, evidenced by shifts in thousands of peak positions post-TBI. Remarkably, these alterations remain largely unaffected by early degradation. Our results robustly endorse the notion that integrating and incorporating these specific loci for injury and disease diagnosis in forensic samples holds tremendous promise for practical application. We further validated the above results using human cortical tissue, which supported that early degradation did not significantly affect chromatin accessibility. This pioneering advancement in molecular diagnostic techniques may revolutionize the field of forensic science, especially forensic pathology.
Asunto(s)
Cromatina , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/diagnóstico , Humanos , Análisis de Secuencia de ADN/métodos , Transposasas/genética , Degradación Necrótica del ADN , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
PURPOSE: To assess the Xpert MTB/XDR assay's efficiency in promptly detecting resistance to isoniazid, fluoroquinolones, ethionamide, and second-line injectable drugs among tuberculosis (TB) patients. METHODS: From August 2020 to July 2021, TB suspected patient samples were enrolled at a tertiary care center for our study. We conducted mycobacterial culture, phenotypic DST using proportion method in liquid culture at WHO-recommended concentrations, and the line probe assay (LPA). Simultaneously, the Index test, Xpert MTB/XDR, was performed following the manufacturer's instructions. RESULTS: Among 360 samples, 107 were excluded due to incomplete information. Resistance to isoniazid, levofloxacin and moxifloxacin was found in 45/251, 21/251 and 20/251 samples, respectively by phenotypic DST. The diagnostic accuracy of Index test, taking phenotypic DST as a reference standard, was 95.8%, 99.04%, and 99.05% for isoniazid, levofloxacin, and moxifloxacin, respectively. The Index test assay demonstrated a specificity of 99.1% for detecting SLID resistance, yielding a diagnostic accuracy of 99.2. Comparing the Index test with LPA revealed a significant enhancement in sensitivity for detecting isoniazid resistance (86.7% vs. 82.2%). CONCLUSIONS: The Index test exhibited promising outcomes in identifying resistance to isoniazid and fluoroquinolones, surpassing the performance of the LPA. This could be valuable for promptly initiating treatment in cases of drug-resistant tuberculosis.