Nanopore-based targeted next-generation sequencing of tissue samples for tuberculosis diagnosis.

Objective: Diagnosing tuberculosis (TB) can be particularly challenging in the absence of sputum for pulmonary tuberculosis cases and extrapulmonary TB (EPTB). This study evaluated the utility of nanopore-based targeted next-generation sequencing (tNGS) for diagnosing TB in tissue samples, and compared its efficacy with other established diagnostic methods. Methods: A total of 110 tissue samples from clinical cases were examined. The sensitivity and specificity of tNGS were benchmarked against a range of existing diagnostic approaches including hematoxylin and eosin (HE) staining in conjunction with acid-fast bacilli (AFB) detection, HE staining combined with PCR, HE staining paired with immunohistochemistry (IHC) using anti-MPT64, and the Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) assay. Results: The sensitivity and specificity of tNGS were 88.2 and 94.1%, respectively. The respective sensitivities for HE staining combined with AFB, HE staining combined with PCR, HE staining combined with IHC using anti-MPT64, and Xpert MTB/RIF were 30.1, 49.5, 47.3, and 59.1%. The specificities for these methods were 82.4, 88.2, 94.1, and 94.1%, respectively. Analysis of drug resistance based on tNGS results indicated that 10 of 93 TB patients (10.75%) had potential drug resistance. Conclusion: Targeted next-generation sequencing achieved higher accuracy than other established diagnostic methods, and can play a crucial role in the rapid and accurate diagnosis of TB, including drug-resistant TB.

Target next-generation sequencing for the accurate diagnosis of Parvimonas micra lung abscess: a case series and literature review.

Background: Parvimonas micra (P. micra) has been identified as a pathogen capable of causing lung abscesses; however, its identification poses challenges due to the specialized culture conditions for anaerobic bacterial isolation. Only a few cases of lung abscesses caused by P. micra infection have been reported. Therefore, we describe the clinical characteristics of lung abscesses due to P. micra based on our case series. Methods: A retrospective analysis was conducted on eight patients who were diagnosed with lung abscesses attributed to P. micra. Detection of P. micra was accomplished through target next-generation sequencing (tNGS). A systematic search of the PubMed database using keywords "lung abscess" and "Parvimonas micra/Peptostreptococcus micros" was performed to review published literature pertaining to similar cases. Results: Among the eight patients reviewed, all exhibited poor oral hygiene, with four presenting with comorbid diabetes. Chest computed tomography (CT) showed high-density mass shadows with necrosis and small cavities in the middle. Bronchoscopic examination revealed purulent sputum and bronchial mucosal inflammation. Thick secretions obstructed the airway, leading to the poor drainage of pus, and the formation of local abscesses leading to irresponsive to antibiotic therapy, which finally protracted recovery time. P. micra was successfully identified in bronchoalveolar lavage fluid (BALF) samples from all eight patients using tNGS; in contrast, sputum and BALF bacterial cultures yielded negative results, with P. micra cultured from only one empyema sample. Following appropriate antibiotic therapy, seven patients recovered. In previously documented cases, favorable outcomes were observed in 77.8% of individuals treated with antibiotics and 22.2% were cured after surgical interventions for P. micra lung abscesses. Conclusions: This study enriches our understanding of the clinical characteristics associated with lung abscesses attributed to P. micra. Importantly, tNGS has emerged as a rapid and effective diagnostic test in scenarios where traditional sputum cultures are negative. Encouragingly, patients with lung abscesses caused by P. micra infection exhibit a favorable prognosis with effective airway clearance and judicious anti-infective management.

Etiological diagnostic performance of probe capture-based targeted next-generation sequencing in bloodstream infection.

Background: A rapid and precise etiological diagnosis is crucial for the effective treatment of bloodstream infection (BSI). In this study, the performance of probe capture-based targeted next-generation sequencing (tNGS) was compared to that of blood culture and metagenomic next-generation sequencing (mNGS) in detecting potential pathogens in patients with BSI. Methods: A total of 80 patients with suspected BSI were prospectively enrolled from 24 November 2023 to 30 December 2023 at Zhongshan Hospital, Shanghai, China. All 80 participants underwent simultaneous blood culture, blood mNGS, and blood tNGS after admission when febrile, and the results were compared. Results: Among the 80 participants, 11 were clinically diagnosed with noninfectious fever, and 69 were diagnosed with BSI. Blood tNGS had a higher sensitivity for the diagnosis of BSI than blood culture (91.3% vs. 23.2%, P<0.001) and blood mNGS (91.3% vs. 69.6%, P=0.001). There was no significant difference in specificity between blood mNGS and tNGS (81.8% vs. 100.0%, P=0.13). Blood tNGS demonstrated a faster turnaround time than blood culture and blood mNGS. In 22 (31.9%) patients with BSI, targeted adjustment of the anti-infectious therapy according to the blood tNGS results resulted in clinical improvement. Conclusions: Blood tNGS may be a promising tool for detecting potential pathogens in patients with BSI. The application of blood tNGS for BSI could guide anti-infectious treatment strategies and might improve clinical outcomes.

Targeted genome sequencing for tuberculosis drug susceptibility testing in South Africa: a proposed diagnostic pipeline.

In July 2023, the World Health Organization (WHO) began recommending targeted next-generation sequencing (tNGS), due to its ability to detect resistance to many drugs with a single test. In March 2023, South Africa further adopted the GeneXpert XDR cartridge, which detects mutations associated with resistance to second-line injectable drugs. Here, we consider the feasibility for implementing tNGS in South Africa, what such a facility might look like and the specific context of this upper-middle-income country. Whilst the WHO now recommends tNGS for TB diagnostics and DST, there are many economic and infrastructural challenges opposing its deployment. In lieu of this, we instead recommend a stratified diagnostic pipeline that utilizes South Africa's existing GeneXpert technologies, attempting to reduce the costs associated with implementation of tNGS.

Diagnostic value of tNGS vs Xpert MTB/RIF in childhood TB.

Objectives: To evaluate the diagnostic value of targeted next generation sequencing (tNGS) in childhood tuberculosis (TB) and compare the accuracy with Xpert MTB/RIF method. Methods: Children aged ≤18 years with symptoms suggestive of TB during July 2021 to December 2022 at Beijing Children's Hospital were included, and the performances of tNGS and Xpert were evaluated. Results: A total of 103 children with suspected TB were recruited, including 72 discharge diagnosis of TB and 31 non-TB cases. The mean age was 7.37 ± 4.77 years, and 62.1 % were male. The most common type of specimens was gastric aspirate (GA) (59, 57.3 %). Among all the 72 TB patients, tNGS showed higher sensitivity than Xpert, but the difference was not significant (34.7 %, 25/72 vs 20.8 %, 15/72; P = 0.063). The specificities of tNGS and Xpert were 87.1 % (27/31) and 96.8 % (30/31), respectively (P = 0.162). Among different types of specimen, the highest sensitivity of tNGS on sputum and pus was observed (80.0 %, 4/5), followed by pleural effusion (50.0 %, 2/4). One rifampin resistance and one protionamide resistance were detected in bacteriologically confirmed TB by tNGS. Conclusion: tNGS had a higher sensitivity but lower specificity compared to Xpert in diagnosis of children TB. tNGS yielded higher sensitivity than Xpert on gastric aspirate and sputum and pus.

Two-Time Multiplexed Targeted Next-Generation Sequencing Might Help the Implementation of Germline Screening Tools for Myelodysplastic Syndromes/Hematologic Neoplasms.

Next-generation sequencing (NGS) tools have importantly helped the classification of myelodysplastic syndromes (MDS), guiding the management of patients. However, new concerns are under debate regarding their implementation in routine clinical practice for the identification of germline predisposition. Cost-effective targeted NGS tools would improve the current standardized studies and genetic counseling. Here, we present our experience in a preliminary study detecting variants using a two-time multiplexed library strategy. Samples from different MDS patients were first mixed before library preparation and later multiplexed for a sequencing run. Two different mixes including a pool of three (3×) and four (4×) samples were evaluated. The filtered variants found in the individually sequenced samples were compared with the variants found in the two-time multiplexed studies to determine the detection efficiency scores. The same candidate variants were found in the two-time multiplexed studies in comparison with the individual tNGS. The variant allele frequency (VAF) values of the candidate variants were also compared. No significant differences were found between the expected and observed VAF percentages in both the 3× (p-value 0.74) and 4× (p-value 0.34) multiplexed studies. Our preliminary results suggest that the two-time multiplexing strategy might have the potential to help reduce the cost of evaluating germline predisposition.

Acute Porphyromonas gingivalis Subdural Abscess with Brain Abscess in the Left Temporal Lobe: A Case Report and Review of Literature.

Background: Brain abscesses are a rare but serious complication of focal intracerebral infection. Case Description: We present a patient of acute subdural abscess with brain abscess in the left temporal lobe. After craniotomy, combined with the Third Next Generation Sequencing and Gene Diagnosis (TNGS & GD) of abscess, we prescribed sensitive antibiotics; the patient recovers well and the abscess did not recur. Conclusion: For patients with acute subdural abscess, combined craniotomy and the TNGS & GD of abscess could achieve good results.

Evaluating the diagnostic accuracy and clinical utility of 16S and 18S rRNA gene targeted next-generation sequencing based on five years of clinical experience.

BACKGROUND: The use of 16S/18S rRNA targeted next-generation sequencing (tNGS) has improved microbial diagnostics, however, the use of tNGS in a routine clinical setting requires further elucidation. We retrospectively evaluated the diagnostic accuracy and clinical utility of 16S/18S tNGS, routinely used in the North Denmark Region between 2017 and 2021. METHODS: We retrieved 544 tNGS results from 491 patients hospitalised with suspected infection (e.g. meningitis, pneumonia, intraabdominal abscess, osteomyelitis and joint infection). The tNGS assays was performed using the Illumina MiSeq desktop sequencer, and BION software for annotation. The patients' diagnosis and clinical management was evaluated by medical chart review. We calculated sensitivity and specificity, and determined the diagnostic accuracy of tNGS by defining results as true positive, true negative, false positive, and false negative. RESULTS: Overall, tNGS had a sensitivity of 56% and a specificity of 97%. tNGS was more frequently true positive compared to culture (32% vs 18%), and tNGS detected a greater variety of bacteria and fungi, and was more frequently polymicrobial. However, the total diagnostic turnaround time was 16 days, and although 73% of tNGS results were true positive or true negative, only 4.4% of results led to changes in clinical management. CONCLUSIONS: As a supplement to culture, tNGS improves identification of pathogenic microorganisms in a broad range of clinical specimens. However, the long turnaround time of tNGS in our setting may have contributed to a limited clinical utility. An improved turnaround time can be the key to improved clinical utility in a future setting.

Detection of Single-Nucleotide and Copy Number Defects Underlying Hyperphenylalaninemia by Next-Generation Sequencing.

Hyperphenylalaninemia (HPA) is the most common inherited amino acid metabolism disorder characterized by serious clinical manifestations, including irreversible brain damage, intellectual deficiency and epilepsy. Due to its extensive genic and allelic heterogeneity, next-generation sequencing (NGS) technology may help to identify the molecular basis of this genetic disease. Herein, we describe the development and validation of a targeted NGS (tNGS) approach for the simultaneous detection of single-nucleotide changes and copy number variations (CNVs) in genes associated with HPA (PAH, GCH1, PTS, QDPR, PCBD1, DNAJC12) or useful for its differential diagnosis (SPR). Our tNGS approach offers the possibility to detail, with a high accuracy and in a single workflow, the combined effect of a broader spectrum of genomic variants in a comprehensive view, providing a significant step forward in the development of optimized patient care and management.

L. pneumophila Infection Diagnosed by tNGS in a Lady with Lymphadenopathy.

We report a case of a 34-year-old lady with multiple joint pain. Autoimmune diseases were considered first with a positive result of anti-Ro antibody and her right knee joint cavity effusion. Later, bilateral interstitial changes in her lungs and mediastinal lymphadenopathy were found after chest CT scanning. Empirical quinolone therapy was given although pathological examinations of blood, sputum and bronchoalveolar lavage fluid (BALF) did not find anything. Finally, Legionella pneumophila was identified by target next-generation sequencing (tNGS) detection. This case highlighted the timely use of tNGS, a new tool with fast speed, high accuracy and effective cost, could help to identify atypical infection and start an early therapy.

Resolution of an insidious and migratory Mycobacterium tuberculosis-associated secondary organizing pneumonia: a case report and literature review.

BACKGROUND: Organizing pneumonia (OP) is a rare interstitial lung disease. Secondary organizing pneumonia (SOP) caused by Mycobacterium tuberculosis (MTB) is extremely rare. Migratory MTB-associated SOP is more deceptive and dangerous. When insidious tuberculosis (TB) is not recognized, SOP would be misdiagnosed as cryptogenic organizing pneumonia (COP). Use of steroid hormone alone leads to the progression of TB foci or even death. Clues of distinguishing atypical TB at the background of OP is urgently needed. CASE PRESENTATION: A 56-year-old female patient was hospitalized into the local hospital because of cough and expectoration for more than half a month. Her medical history and family history showed no relation to TB or other lung diseases. Community-acquired pneumonia was diagnosed and anti-infection therapy was initialized but invalid. The patient suffered from continuous weigh loss. More puzzling, the lesions were migratory based on the chest computed tomography (CT) images. The patient was then transferred to our hospital. The immunological indexes of infection in blood and pathogenic tests in sputum and the bronchoalveolar lavage fluid were negative. The percutaneous lung puncture biopsy and pathological observation confirmed OP, but without granulomatous lesions. Additionally, pathogen detection of the punctured lung tissues by metagenomics next generation sequencing test (mNGS) were all negative. COP was highly suspected. Fortunately, the targeted next-generation sequencing (tNGS) detected MTB in the punctured lung tissues and MTB-associated SOP was definitely diagnosed. The combined therapy of anti-TB and prednisone was administrated. After treatment for 10 days, the partial lesions were significantly resorbed and the patient was discharged. In the follow-up of half a year, the patient was healthy. CONCLUSIONS: It is difficult to distinguish SOP from COP in clinical practice. Diagnosis of COP must be very cautious. Transient small nodules and cavities in the early lung image are a clue to consider TB, even though all pathogen tests are negative. tNGS is also a powerful tool to detect pathogen, ensuring prompt diagnosis of TB-related SOP. For clinicians in TB high burden countries, we encourage them to keep TB in mind before making a final diagnosis of COP.

Operationalising targeted next-generation sequencing for routine diagnosis of drug-resistant TB.

BACKGROUND: Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India. METHODS: Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below. RESULTS: Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119. CONCLUSIONS: Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.

CONTEXTE: Les tests phénotypiques de sensibilité aux médicaments (pDST) pour Mycobacterium tuberculosis peuvent prendre jusqu'à 8 semaines, tandis que les tests moléculaires conventionnels identifient un ensemble limité de mutations de résistance. Le séquençage ciblé de la prochaine génération (tNGS) offre des résultats rapides pour prédire la résistance globale aux médicaments, et cette étude avait pour objectif d'explorer sa faisabilité opérationnelle au sein d'un laboratoire de santé publique à Mumbai, en Inde. MÉTHODES: Des échantillons pulmonaires de patients consentants testés positifs au Xpert MTB ont été testés pour la résistance aux médicaments par des méthodes conventionnelles et en utilisant le tNGS. Les expériences des membres de l'équipe de l'étude en matière de fonctionnement du laboratoire et de mise en œuvre logistique sont présentées ci-dessous. RÉSULTATS: Sur le nombre total de patients testés, 70% (113/161) n'avaient pas d'antécédents de TB ou de traitement ; cependant, 88,2% (n = 142) présentaient une TB résistante à la rifampicine/multirésistante aux médicaments (RR/MDR-TB). La concordance entre les prédictions de résistance de la tNGS et de la pDST était élevée pour la plupart des médicaments, la tNGS identifiant globalement la résistance avec plus de précision. La tNGS a été intégrée et adaptée au flux de travail du laboratoire ; toutefois, la mise en lots des échantillons a entraîné un délai d'obtention des résultats beaucoup plus long, le plus rapide étant de 24 jours. L'extraction manuelle de l'ADN a été source d'inefficacité ; le protocole a donc été optimisé. L'analyse des mutations non caractérisées et l'interprétation des modèles de rapport ont nécessité une expertise technique. Le coût du tNGS par échantillon s'élevait à US$230, contre US$119 pour le pDST. CONCLUSIONS: La mise en œuvre de la tNGS est possible dans les laboratoires de référence. Elle permet d'identifier rapidement la résistance aux médicaments et devrait être considérée comme une alternative potentielle à la pDST.

An odyssey from laboratory to field ? - Portable tNGS system for TB diagnosis in programmatic setting.

In spite of the elaborate diagnostic modalities available in India, there are certain shortcomings which will affect patient management. In order to address the gaps, NTEP offers scope for whole genome sequencing at few of its reference laboratories. Next generation sequencing comprising of whole genome sequencing (WGS) and targeted next generation sequencing (tNGS) are upcoming fields in TB diagnosis In a programmatic setting, tNGS offers great promise for smear positive or NAAT positive samples to be used with a Minion platform in a field setting beyond just the National reference laboratories. Once materialised, the tNGS would offer personalised patient management as well as help in public health by identification of outbreaks, transmission chain monitoring and drug resistance surveillance.

Modelling polycystic liver disease progression using age-adjusted liver volumes and targeted mutational analysis.

Background & Aims: Polycystic liver disease (PLD) manifests as numerous fluid-filled cysts scattered throughout the liver parenchyma. PLD most commonly develops in females, either as an extra-renal manifestation of autosomal-dominant polycystic kidney disease (ADPKD) or as isolated autosomal-dominant polycystic liver disease (ADPLD). Despite known genetic causes, clinical variability challenges patient counselling and timely risk prediction is hampered by a lack of genotype-phenotype correlations and prognostic imaging classifications. Methods: We performed targeted next-generation sequencing and multiplex ligation-dependent probe amplification to identify the underlying genetic defect in a cohort of 80 deeply characterized patients with PLD. Identified genotypes were correlated with total liver and kidney volume (assessed by CT or MRI), organ function, co-morbidities, and clinical endpoints. Results: Monoallelic diagnostic variants were identified in 60 (75%) patients, 38 (48%) of which pertained to ADPKD-gene variants (PKD1, PKD2, GANAB) and 22 (27%) to ADPLD-gene variants (PRKCSH, SEC63). Disease severity defined by age at waitlisting for liver transplantation and first PLD-related hospitalization was significantly more pronounced in mutation carriers compared to patients without genetic diagnoses. While current imaging classifications proved unable to differentiate between severe and moderate courses, grouping by estimated age-adjusted total liver volume progression yielded significant risk discrimination. Conclusion: This study underlines the predictive value of providing a molecular diagnosis for patients with PLD. In addition, we propose a novel risk-classification model based on age- and height-adjusted total liver volume that could improve individual prognostication and personalized clinical management. Lay summary: Polycystic liver disease (PLD) is a highly variable condition that can be asymptomatic or severe. However, it is currently difficult to predict clinical outcomes such as hospitalization, symptom burden, and need for transplantation in individual patients. In the current study, we aimed to investigate the clinical value of genetic confirmation and an age-adjusted total liver volume classification for individual disease prediction. While genetic confirmation generally pointed to more severe disease, estimated age-adjusted increases in liver volume could be useful for predicting clinical outcomes.

Central nervous system aspergillosis misdiagnosed as Toxoplasma gondii encephalitis in a patient with AIDS: a case report.

BACKGROUND: Patients with acquired immunodeficiency syndrome (AIDS) tend to suffer from several central nervous system (CNS) infections due to hypoimmunity. However, CNS aspergillosis (CNSAG) is extremely rare and difficult to diagnose. Thus, it is easily misdiagnosed. CASE PRESENTATION: We reported a 47-year-old male AIDS patient with ghosting vision and anhidrosis on the left head and face. He was accordingly diagnosed with Toxoplasma gondii encephalitis (TE) at other hospitals, for which he received regular anti-Toxoplasma gondii and anti-human immunodeficiency virus (anti-HIV) treatment. Then, the patient was transferred to our hospital due to a lack of any improvement with the prescribed treatment. The patient's neurological examination revealed no abnormalities at admission, only a slight change in the cerebrospinal fluid. His cranial magnetic resonance imaging (MRI) revealed multiple abnormal signals in the brain parenchyma, and his blood was positive for Toxoplasma gondii IgG antibody. The initial diagnosis at our hospital was also TE. Considering the poor efficacy of anti-TE treatment, cerebrospinal fluid metagenomics next-generation sequencing (mNGS) was performed, but no pathogenic bacteria were detected. However, Aspergillus fumigatus was detected in the cerebrospinal fluid via targeted next-generation sequencing (tNGS) and bronchoalveolar alveolar lavage fluid via mNGS. The diagnosis was accordingly revised to CNSAG combined with his other clinical manifestations. After administering voriconazole antifungal therapy, the patient's symptoms were relieved, with improved absorption of the intracranial lesions. CONCLUSIONS: The present case experience indicates the need for clinicians to strengthen their understanding of CNSAG. Moreover, for patients with diagnostic difficulties, early mNGS and tNGS (using biological samples with only a few pathogens) are helpful for early diagnosis and treatment, potentially allowing patients to achieve favorable outcomes.

Detection of Structural Variants by NGS: Revealing Missing Alleles in Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are a heterogeneous group of rare multisystem metabolic disorders occurring mostly in infancy and childhood, characterized by a gradual accumulation of non-degraded substrates inside the cells. Although biochemical enzymatic assays are considered the gold standard for diagnosis of symptomatic patients, genotyping is a requirement for inclusion in enzyme replacement programs and is a prerequisite for carrier tests in relatives and DNA-based prenatal diagnosis. The emerging next-generation sequencing (NGS) technologies are now offering a powerful diagnostic tool for genotyping LSDs patients by providing faster, cheaper, and higher-resolution testing options, and are allowing to unravel, in a single integrated workflow SNVs, small insertions and deletions (indels), as well as major structural variations (SVs) responsible for the pathology. Here, we summarize the current knowledge about the most recurrent and private SVs involving LSDs-related genes, review advantages and drawbacks related to the use of the NGS in the SVs detection, and discuss the challenges to bring this type of analysis in clinical diagnostics.

Molecular Profiles of Advanced Urological Cancers in the PERMED-01 Precision Medicine Clinical Trial.

INTRODUCTION: The prognosis of advanced urological cancers (AUC) remains unfavorable, and few data are available regarding precision medicine. METHODS: the PERMED-01 prospective clinical trial assessed the impact of molecular profiling in adults with refractory advanced solid cancer, in terms of number of patients with tumor actionable genetic alterations (AGA), feasibility, description of molecular alterations, treatment, and clinical outcome. We present here those results in the 64 patients enrolled with AUC. DNA extracted from a new tumor biopsy was profiled in real-time (targeted NGS, whole-genome array-comparative genomic hybridization), and the results were discussed during a weekly molecular tumor board meeting. RESULTS: a complete molecular profile was obtained in 49 patients (77%). Thirty-eight (59%) had at least one AGA. Twelve (19%) received a matched therapy on progression, of which 42% had a PFS2/PFS1 ratio ≥ 1.3 versus 5% in the "non-matched therapy group" (n = 25). The objective response and disease control rates were higher in the "matched therapy group" (33% and 58%, respectively) than in the "non-matched therapy group" (13% and 22%), as was the 6-month OS (75% vs. 42%). CONCLUSION: the profiling of a newly biopsied tumor sample identified AGA in 59% of patients with AUC, led to "matched therapy" in 19%, and provided clinical benefit in 8%.

Potential sensing of toxic chemical warfare agents (CWAs) by twisted nanographenes: A first principle approach.

The toxic chemical warfare agents (CWAs) are extremely harmful to the living organisms. Their efficient detection and removal in a limited time span are essential for the human health and environmental security. Twisted nanographenes have great applications in the fields of energy storage and optoelectronics, but their use as sensors is rarely described. Therefore, we have explored the sensitivity and selectivity of twisted nanographene analogues (C32H16, C64H32) towards selected toxic CWAs, including phosgene, thiophosgene and formaldehyde. The interaction between CWAs and twisted nanographenes is mainly interpreted by considering the optimized geometries, adsorption energies, natural bond orbital (NBO), frontier molecular orbital (FMO), non-covalent interaction (NCI) and quantum theory of atoms in molecules (QTAIM) analyses. The structural geometries show that the central octagon of twisted nanographenes is the most favorable site of interaction. The interaction energies reveal the physisorption of selected CWAs on tNGs surface. The average energy gap change (%EH-La) and % sensitivity are quantitatively determined to evaluate the sensing capability of the twisted nanographenes. Among the selected CWAs molecules, the sensitivity of tNG analogues (C32H16 and C64H32) is superior towards thiophosgene (ThP), which is revealed by the high interaction energies of -8.19 and - 12.17 kcal/mol, respectively. This theoretical study will help experimentalists to devise novel sensors based on twisted nanographenes for the detection of toxic CWAs which may also work efficiently under the humid conditions.

Implementation of Hospital-Based Supplemental Duchenne Muscular Dystrophy Newborn Screening (sDMDNBS): A Pathway to Broadening Adoption.

Duchenne muscular dystrophy (DMD) is not currently part of mandatory newborn screening, despite the availability of a test since 1975. In the absence of screening, a DMD diagnosis is often not established in patients until 3-6 years of age. During this time, irreversible muscle degeneration takes place, and clinicians agree that the earlier therapy is initiated, the better the long-term outcome. With recent availability of FDA-approved DMD therapies, interest has renewed for adoption by state public health programs, but such implementation is a multiyear process. To speed access to approved therapies, we implemented a unique, hospital-based program offering parents of newborns an optional, supplemental DMD newborn screen (NBS) via a two-tiered approach: utilizing a creatine kinase (CK) enzyme assay coupled with rapid targeted next-generation sequencing (tNGS) for the DMD gene (using a Whole-Exome Sequencing (WES) assay). The tNGS/WES assay integrates the ability to detect both point mutations and large deletion/duplication events. This tiered newborn screening approach allows for the opportunity to improve treatment and outcomes, avoid the diagnostic delays, and diminish healthcare disparities. To implement this screening algorithm through hospitals in a way that would ultimately be acceptable to public health laboratories, we chose an FDA-approved CK-MM immunoassay to avoid the risks of false-negative/-positive results. Because newborn CK values can be affected due to non-DMD-related causes such as birth trauma, a confirmatory repeat CK assay on a later dried blood spot (DBS) collection has been proposed. Difficulties associated with non-routine repeat DBS collection, including the tracking and recall of families, and the potential creation of parental anxiety associated with false-positive results, can be avoided with this algorithm. Whereas a DMD diagnosis is essentially ruled out by the absence of detected DMD sequence abnormalities, a subsequent CK would still be warranted to confirm resolution of the initial elevation, and thus the absence of non-DMD muscular dystrophy or other pathologies. To date, we have screened over 1500 newborns (uptake rate of ~80%) by a CK-MM assay, and reflexed DMD tNGS in 29 of those babies. We expect the experience from this screening effort will serve as a model that will allow further expansion to other hospital systems until a universal public health screening is established.

A Comprehensive, Targeted NGS Approach to Assessing Molecular Diagnosis of Lysosomal Storage Diseases.

With over 60 different disorders and a combined incidence occurring in 1:5000-7000 live births, lysosomal storage diseases (LSDs) represent a major public health problem and constitute an enormous burden for affected individuals and their families. Several reasons make the diagnosis of LSDs an arduous task for clinicians, including the phenotype and penetrance variability, the shared signs and symptoms, and the uncertainties related to biochemical enzymatic assay results. Developing a powerful diagnostic tool based on next generation sequencing (NGS) technology may help reduce the delayed diagnostic process for these families, leading to better outcomes for current therapies and providing the basis for more appropriate genetic counseling. Herein, we employed a targeted NGS-based panel to scan the coding regions of 65 LSD-causative genes. A reference group sample (n = 26) with previously known genetic mutations was used to test and validate the entire workflow. Our approach demonstrated elevated analytical accuracy, sensitivity, and specificity. We believe the adoption of comprehensive targeted sequencing strategies into a routine diagnostic route may accelerate both the identification and management of LSDs with overlapping clinical profiles, producing a significant reduction in delayed diagnostic response with beneficial results in the treatment outcome.