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GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre-extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.

Bioinformatics was established at the IMGGE in collaboration with the Beijing Genomics Institute (BGI) and with the support of the Government of the Republic of Serbia. The Center was founded with the aim of accelerating the implementation of 4P medicine (Preventive, Predictive, Personalized, and Participatory), in our country by using the cutting-edge tools of molecular biology and information technology. As such, the center is unique in Serbia and the South East Europe region. The state-of-the-art equipment existing at the Center (DNBSEQ-G400 (BGI), NextSeq 550Dx, 2000 and MiSeq (Illumina) Sequencing Systems; MinION (OxfordNanopore); MGISP-9600 High-throughput Automated Sample Preparation System) offered a wide range of application: the whole genome and whole exome sequencing, targeted sequencing, transcriptome sequencing and more. The analysis of data was facilitated by the access to the National Platform for Artificial Intelligence providing space for secure data storage, and to a supercomputer (Nvidia), critical for processing and analyzing Big Data. Since its establishment more than 2000 SARS-CoV2 genomes were sequenced, 300 patient samples undervent diagnostic work-up. Pilot project for NIFTYPro screening was finished encompasing 58 pregnant women. First transcriptomes and metagenomes were analyzed. Further implementation of NGS methodology in research and for diagnostics, and intensive development of bioinformatics, by strengthening the hardware and software capacities and the education of the bioinformatics team, will lead us in becoming driving force of the development of biomedicine and biotechnology in Serbia, extending collaboration beyond our borders.

Precision cancer medicine is a multidisciplinary team effort that requires involvement and commitment of many stakeholders including the society at large. Building on the success of significant advances in precision therapy for oncological patients over the last two decades, future developments will be significantly shaped by improvements in scalable molecular diagnostics in which increasingly complex multilayered datasets require transformation into clinically useful information guiding patient management at fast turnaround times. Adaptive profiling strategies involving tissue- and liquid-based testing that account for the immense plasticity of cancer during the patient's journey and also include early detection approaches are already finding their way into clinical routine and will become paramount. A second major driver is the development of smart clinical trials and trial concepts which, complemented by real-world evidence, rapidly broaden the spectrum of therapeutic options. Tight coordination with regulatory agencies and health technology assessment bodies is crucial in this context. Multicentric networks operating nationally and internationally are key in implementing precision oncology in clinical practice and support developing and improving the ecosystem and framework needed to turn invocation into benefits for patients. The review provides an overview of the diagnostic tools, innovative clinical studies, and collaborative efforts needed to realize precision cancer medicine.

Precision oncology has come a long way since the introduction of the first targeted drug (trastuzumab) in 1999. Broad molecular testing of tumor tissue has vastly expanded our knowledge of the biology of cancer, leading to a steep increase in the number of approved targeted drugs and an expansion of the labeled indications of these drugs. Off-label use of these new classes of targeted drugs is nowadays better documented and often performed in clinical trials to maximize the learning potential of these experimental treatments for the medical community. As long as no “cure for cancer” exists, there will be room for improvement of our knowledge and approach to treating patients with cancer. General improvements in the logistics, availability of targeted drugs and access to diagnostics and expertise will likely have the greatest impact on direct benefit for patients. In the future, standardized processing and conservation of tumor tissue/biopsies should be possible in all healthcare facilities, and collaborations and sharing of knowledge and resources with the academic institutes will be viable to delivering precision oncology to all patients. If these conditions are met, more patients may potentially benefit from the knowledge and new treatment options resulting from the precision oncology trials. Also, medical oncologists may learn more about molecular testing and interpreting test results from participation in MTBs. To maximize the impact of precision oncology, international collaborations are of utmost importance and research groups throughout the world are encouraged to share best practices and creative solutions to overcome the hurdles that still hamper new initiatives in the field today. Future clinical research may focus on prospective therapy selection using molecular information from other –omics fields, such as phosphoproteomics, especially in patients where no clear monogenetic driver mutations is identified and a comprehensive pathway analysi

Tuta absoluta and Drosophila suzukii are two agricultural pest insects that have both rapidly spreadworldwide from their original ecosystems. Both cause serious economic losses, with the South American native T. absoluta targeting tomato agriculture and the Asian native D. suzukii targeting soft berry fruits. To inform current management strategies and prevent further introduction of these species, it is necessary to gain insights into current population structure and migration history of these species. Additionally, as T. absoluta has yet to be detected in North America, effective molecular diagnostics are needed to improve quarantine and monitoring efforts. In chapter one, we sequenced whole genomes of hundreds of D. suzukii samples collected worldwide. We identified two major population clusters in the United States and found that West coast D. suzukii populations originated from a combination of migration events from Hawaii and Asia. We saw no strong loss in genetic diversity in invasive populations relative to Asian populations, suggesting ongoing migration is alleviating any bottleneck effects. In chapter two, we sequenced dozens of whole genomes of T. absoluta across Latin America and Spain and found three populations in Latin America. Using population simulation approaches, we found that these populations diverged prior to human agriculture of tomatoes. Additionally, we detected signals of selective sweeps near genes relevant to insecticide resistance and metabolism. In chapter three, we developed two molecular diagnostics to distinguish T. absoluta from two morphologically similar species already present in the United States. The probebased quantitative PCR diagnostic can differentiate between T. absoluta, Phthorimaea operculella, and Keiferia lycopersicella using a thermocycler, while the RPA-Cas12a diagnostic can identity presence of T. absoluta in an isothermal reaction with minimal lab equipment requirements. We expect that these analyses and genomic re

Diagnostic development must occur in parallel with drug development to ensure the longevity of new treatment compounds. Despite an increasing number of novel and repurposed anti-tuberculosis compounds and regimens, there remains a large number of drugs for which no rapid and accurate molecular diagnostic option exists. The lack of rapid drug susceptibility testing for linezolid, bedaquiline, clofazimine, the nitroimidazoles (i.e pretomanid and delamanid) and pyrazinamide at any level of the healthcare system compromises the effectiveness of current tuberculosis and drug-resistant tuberculosis treatment regimens. In the context of current WHO tuberculosis treatment guidelines as well as promising new regimens, we identify the key diagnostic gaps for initial and follow-on tests to diagnose emerging drug resistance and aid in regimen selection. Additionally, we comment on potential gene targets for inclusion in rapid molecular drug susceptibility assays and sequencing assays for novel and repurposed drug compounds currently prioritized in current regimens, and evaluate the feasibility of mutation detection given the design of existing technologies. Based on current knowledge, we also propose design priorities for next generation molecular assays to support triage of tuberculosis patients to appropriate and effective treatment regimens. We encourage assay developers to prioritize development of these key molecular assays and support the continued evolution, uptake, and utility of sequencing to build knowledge of tuberculosis resistance mechanisms and further inform rapid treatment decisions in order to curb resistance to critical drugs in current regimens and achieve End TB targets.Trial registration: ClinicalTrials.gov identifier: NCT05117788..

Sexually transmitted infections (STIs) are and will continue to be a serious public health problem throughout the world according to WHO data, with the aggravating factor that most cases are asymptomatic and, furthermore, there is no other reservoir other than humans. The diagnosis can be made with traditional and molecular tests, the latter include the polymerase chain reaction (PCR), of which there are several types, among them, multiplex PCR that has the capacity to detect polymicrobial STIs from a single sample. The objective of this study was to establish which were the most frequent sexually transmitted infections in different groups of patients, as well as to determine the usefulness of the multiplex PCR technique in the diagnosis of STIs. Methodology. This is an observational, cross-sectional study carried out between 2021 and 2022 with patients who attended the VID Clinical Laboratory for suspected STIs. The collected samples were evaluated using a commercial test based on the multiplex PCR technique and hybridization. The samples processed were: urine and swabs from endocervix, urethra, rectum, pharynx, and ulcers. Results. The study included 1,027 patients, of these, 228 (22.2%) were positive for different sexually transmitted agents, distributed as follows: 50 (21.9%) women, 129 (56.6%) heterosexual men and 49 (21.5%) men who had sex with men (MSM). The average age of the women was 30 years, and that of both groups of men was 36 years. The microorganisms most frequently identified in women were: C. trachomatis (A-K) in 28.6%, followed by herpes simplex virus type 2 (HSV-2) in 26.8% and N. gonorrhoeae in 17.9%. In heterosexual men they were C. trachomatis (A-K) in 37.5%, N. gonorrhoeae in 21.5% and HSV-2 in 18.7%. In MSM they were C. trachomatis (L1-L3) in 32.7%, followed by N. gonorrhoeae in 27.6%, and C. trachomatis (A-K) and HSV-2, both in 13.8%. Polymicrobial infection was identified in 11 heterosexual men, 8 MSM, and 6 women. Conclusion. C. trachomat, Las infecciones de transmisión sexual (ITS) son y seguirán siendo un serio problema de salud pública en todo el mundo según los datos de la OMS, con el agravante que la mayoría de los casos son asintomáticos y, además, no existe otro reservorio distinto al humano. El diagnóstico se puede realizar con pruebas tradicionales y moleculares, estas últimas incluyen la reacción en cadena de la polimerasa (PCR), de las cuales existen varios tipos, entre ellas, la PCR múltiple que tiene la capacidad de detectar ITS polimicrobianas a partir de una sola muestra. El objetivo de este estudio fue establecer cuáles fueron las infecciones de transmisión sexual más frecuentes en diferentes grupos de pacientes, así como determinar la utilidad del uso de la técnica de PCR múltiple en el diagnóstico de las ITS. Metodología. Se trata de un estudio observacional de corte transversal realizado entre los años 2021 y 2022 con pacientes que acudieron al servicio de diagnóstico del Laboratorio Clínico VID por sospecha de ITS. Las muestras recolectadas fueron evaluadas utilizando una prueba comercial basada en la técnica de PCR múltiple e hibridación. Las muestras procesadas fueron: orina e hisopados de endocérvix, uretra, recto, faringe y úlceras. Resultados. Se estudiaron 1.027 pacientes, de estos, 228 (22,2 %) fueron positivos para diferentes agentes de trasmisión sexual, distribuidos así: 50 (21,9 %) mujeres, 129 (56,6 %) hombres heterosexuales y 49 (21,5 %) hombres que tenían sexo con hombres (HSH). La edad promedio de las mujeres fue 30 años, y la de ambos grupos de hombres fue 36 años. Los microorganismos más frecuentemente identificados en mujeres fueron: C. trachomatis (A-K) en 28,6 %, seguido de virus herpes simplex tipo 2 (VHS-2) en 26,8 % y N. gonorrhoeae en 17,9 %. En hombres heterosexuales fueron C. trachomatis (A-K) en 37,5 %, N. gonorrhoeae en 21,5 % y VHS-2 en 18,7 %. En HSH fueron C. trachomatis (L1-L3) en 32,7 %, seguido de N. gonorrhoeae en 27,6 %, y de C. trachomatis (A-K)

Malaria case investigation and reactive case detection (RACD) activities are widely implemented in low transmission settings to identify additional malaria infections and gather surveillance information, but with varying degrees of success. Challenges in conducting RACD include poor diagnostic sensitivity (particularly for low density and asymptomatic infections), knowledge gaps among those conducting RACD, financial and resource constraints, and operational and logistical difficulties. To improve infection detection and better target individuals at highest risk for infection, RACD strategies need to be evaluated and optimized to provide quality and nuanced surveillance information. To support more effective surveillance and response strategies, this PhD project focused on evaluating RACD strategies to improve and optimize malaria surveillance in low transmission settings in the Asia Pacific region. Using a standardized monitoring and evaluation (M&E) tool, case investigation and RACD indicators were assessed, including the knowledge and practices of the staff conducting RACD. This PhD project explored the utility of molecular diagnostics and genotyping and targeted sociobehavorial RACD strategies for increasing infection detection and to understand the relatedness of infections identified during RACD. Also, the acceptability and feasibility of a presumptive treatment-based strategy to reduce malaria (referred to as reactive drug administration (RDA)) was evaluated. Results revealed gaps in case investigation and RACD reporting completeness and timeliness and that staff were not always equipped with the appropriate documentation or have accurate knowledge on how to conduct RACD. Molecular diagnostics used in RACD in Thailand identified an additional 12 (0.6%) infections compared to no RACD-identified infections detected by microscopy. Of the four confirmed infections, only one (25%) was genetically related to the index case. In Indonesia, a sociobehavorial RACD

Malaria case investigation and reactive case detection (RACD) activities are widely implemented in low transmission settings to identify additional malaria infections and gather surveillance information, but with varying degrees of success. Challenges in conducting RACD include poor diagnostic sensitivity (particularly for low density and asymptomatic infections), knowledge gaps among those conducting RACD, financial and resource constraints, and operational and logistical difficulties. To improve infection detection and better target individuals at highest risk for infection, RACD strategies need to be evaluated and optimized to provide quality and nuanced surveillance information. To support more effective surveillance and response strategies, this PhD project focused on evaluating RACD strategies to improve and optimize malaria surveillance in low transmission settings in the Asia Pacific region. Using a standardized monitoring and evaluation (M&E) tool, case investigation and RACD indicators were assessed, including the knowledge and practices of the staff conducting RACD. This PhD project explored the utility of molecular diagnostics and genotyping and targeted sociobehavorial RACD strategies for increasing infection detection and to understand the relatedness of infections identified during RACD. Also, the acceptability and feasibility of a presumptive treatment-based strategy to reduce malaria (referred to as reactive drug administration (RDA)) was evaluated. Results revealed gaps in case investigation and RACD reporting completeness and timeliness and that staff were not always equipped with the appropriate documentation or have accurate knowledge on how to conduct RACD. Molecular diagnostics used in RACD in Thailand identified an additional 12 (0.6%) infections compared to no RACD-identified infections detected by microscopy. Of the four confirmed infections, only one (25%) was genetically related to the index case. In Indonesia, a sociobehavorial RACD

Malaria case investigation and reactive case detection (RACD) activities are widely implemented in low transmission settings to identify additional malaria infections and gather surveillance information, but with varying degrees of success. Challenges in conducting RACD include poor diagnostic sensitivity (particularly for low density and asymptomatic infections), knowledge gaps among those conducting RACD, financial and resource constraints, and operational and logistical difficulties. To improve infection detection and better target individuals at highest risk for infection, RACD strategies need to be evaluated and optimized to provide quality and nuanced surveillance information. To support more effective surveillance and response strategies, this PhD project focused on evaluating RACD strategies to improve and optimize malaria surveillance in low transmission settings in the Asia Pacific region. Using a standardized monitoring and evaluation (M&E) tool, case investigation and RACD indicators were assessed, including the knowledge and practices of the staff conducting RACD. This PhD project explored the utility of molecular diagnostics and genotyping and targeted sociobehavorial RACD strategies for increasing infection detection and to understand the relatedness of infections identified during RACD. Also, the acceptability and feasibility of a presumptive treatment-based strategy to reduce malaria (referred to as reactive drug administration (RDA)) was evaluated. Results revealed gaps in case investigation and RACD reporting completeness and timeliness and that staff were not always equipped with the appropriate documentation or have accurate knowledge on how to conduct RACD. Molecular diagnostics used in RACD in Thailand identified an additional 12 (0.6%) infections compared to no RACD-identified infections detected by microscopy. Of the four confirmed infections, only one (25%) was genetically related to the index case. In Indonesia, a sociobehavorial RACD

The intent of this review is to present basic genetic concepts key to understanding oncogenesis and the role receptor tyrosine kinase (RTK) inhibition plays in the targeted treatment of many cancer types. Oncogenic signaling by RTKs can result from genetic events such as point mutations, chromosomal rearrangements, structural variation, and gene amplification in the cancer genome. The cancer pharmacogenes discussed encode RTKs that exemplify the link between gene variation, the oncogenic process, and the basis of targeted approaches to treatment. Biochemical pathways often involved in oncogenesis and affected by RTK variation are reviewed. Molecular diagnostic testing for the presence of specific gene variants, alterations, and amplifications direct therapy to indicated tyrosine kinase inhibitors and monoclonal antibody drugs. As pharmacists are integral to the selection, preparation, and monitoring of chemotherapy, it is important that they understand the genetic basis for targeted therapies as well as the underlying disease process.

Malaria case investigation and reactive case detection (RACD) activities are widely implemented in low transmission settings to identify additional malaria infections and gather surveillance information, but with varying degrees of success. Challenges in conducting RACD include poor diagnostic sensitivity (particularly for low density and asymptomatic infections), knowledge gaps among those conducting RACD, financial and resource constraints, and operational and logistical difficulties. To improve infection detection and better target individuals at highest risk for infection, RACD strategies need to be evaluated and optimized to provide quality and nuanced surveillance information. To support more effective surveillance and response strategies, this PhD project focused on evaluating RACD strategies to improve and optimize malaria surveillance in low transmission settings in the Asia Pacific region. Using a standardized monitoring and evaluation (M&E) tool, case investigation and RACD indicators were assessed, including the knowledge and practices of the staff conducting RACD. This PhD project explored the utility of molecular diagnostics and genotyping and targeted sociobehavorial RACD strategies for increasing infection detection and to understand the relatedness of infections identified during RACD. Also, the acceptability and feasibility of a presumptive treatment-based strategy to reduce malaria (referred to as reactive drug administration (RDA)) was evaluated. Results revealed gaps in case investigation and RACD reporting completeness and timeliness and that staff were not always equipped with the appropriate documentation or have accurate knowledge on how to conduct RACD. Molecular diagnostics used in RACD in Thailand identified an additional 12 (0.6%) infections compared to no RACD-identified infections detected by microscopy. Of the four confirmed infections, only one (25%) was genetically related to the index case. In Indonesia, a sociobehavorial RACD

[ES] La enfermedad de Wilson (EW) es un trastorno hereditario del metabolismo del cobre causado por mutaciones en ATP7B, que codifica una proteína transportadora de cobre en el hígado. Su mal funcionamiento provoca un fallo en la excreción biliar de cobre y una acumulación progresiva de este metal en el organismo, especialmente en hígado y cerebro. En este trabajo, se explora la posible utilidad de miRNAs circulantes en plasma para identificar biomarcadores que sirvan para controlar la progresión de la enfermedad en pacientes con EW bajo tratamiento. Los modelos desarrollados para cada miRNA mostraron un buen rendimiento al clasificar a los pacientes con factores de evolución desfavorable, por lo que estos tres miRNAs se proponen como candidatos para mejorar el seguimiento clínico o para respaldar la eficacia de nuevas terapias en la EW., [CA] La malaltia de Wilson és un trastorn hereditari del metabolisme del coure causat per mutacions en ATP7B, que codifica per a una proteïna transportadora del coure al fetge. El seu mal funcionament produeix alteracions en l'excreció biliar i l'acumulació progressiva de coure, especialment en fetge i cervell. Es va explorar la possible utilitat del perfil de miRNAs circulants com biomarcadors de progressió de la patologia hepàtica. L'avaluació dels models obtinguts per a cadascun dels tres miRNAs va mostrar un bon rendiment per a classificar al grup de pacients amb factors d’evolució desfavorable, en conseqüència, es proposen com a candidats per tal de millorar el seguiment clínic o comprovar l’efectivitat de noves teràpies en la malaltia de Wilson., [EN] Wilson disease (WD) is an inherited disorder of copper metabolism caused by mutations in ATP7B, which encodes for a liver copper-transporting protein. Its dysfunction causes a deficit in biliary copper excretion and a progressive accumulation of this metal in the organism, mainly in liver and brain. In this work, circulating miRNAs profiling in plasma has been accomplished to identify biomarkers that could serve to monitor disease progression in WD patients under chelation therapy. Developed models for each miRNA exhibited good performance classifying patients with poor outcome factors, consequently, these three miRNAs are proposed as candidates to improve clinical follow-up or to support efficacy of novel therapies in WD.

[ES] La enfermedad de Wilson (EW) es un trastorno hereditario del metabolismo del cobre causado por mutaciones en ATP7B, que codifica una proteína transportadora de cobre en el hígado. Su mal funcionamiento provoca un fallo en la excreción biliar de cobre y una acumulación progresiva de este metal en el organismo, especialmente en hígado y cerebro. En este trabajo, se explora la posible utilidad de miRNAs circulantes en plasma para identificar biomarcadores que sirvan para controlar la progresión de la enfermedad en pacientes con EW bajo tratamiento. Los modelos desarrollados para cada miRNA mostraron un buen rendimiento al clasificar a los pacientes con factores de evolución desfavorable, por lo que estos tres miRNAs se proponen como candidatos para mejorar el seguimiento clínico o para respaldar la eficacia de nuevas terapias en la EW., [CA] La malaltia de Wilson és un trastorn hereditari del metabolisme del coure causat per mutacions en ATP7B, que codifica per a una proteïna transportadora del coure al fetge. El seu mal funcionament produeix alteracions en l'excreció biliar i l'acumulació progressiva de coure, especialment en fetge i cervell. Es va explorar la possible utilitat del perfil de miRNAs circulants com biomarcadors de progressió de la patologia hepàtica. L'avaluació dels models obtinguts per a cadascun dels tres miRNAs va mostrar un bon rendiment per a classificar al grup de pacients amb factors d’evolució desfavorable, en conseqüència, es proposen com a candidats per tal de millorar el seguiment clínic o comprovar l’efectivitat de noves teràpies en la malaltia de Wilson., [EN] Wilson disease (WD) is an inherited disorder of copper metabolism caused by mutations in ATP7B, which encodes for a liver copper-transporting protein. Its dysfunction causes a deficit in biliary copper excretion and a progressive accumulation of this metal in the organism, mainly in liver and brain. In this work, circulating miRNAs profiling in plasma has been accomplished to identify biomarkers that could serve to monitor disease progression in WD patients under chelation therapy. Developed models for each miRNA exhibited good performance classifying patients with poor outcome factors, consequently, these three miRNAs are proposed as candidates to improve clinical follow-up or to support efficacy of novel therapies in WD.

Research using nucleic acid amplification tests (NAATs) have repeatedly found rectal and oropharyngeal infections with Chlamydia trachomatis and Neisseria gonorrhoeae to be common and potentially more difficult to treat than genital infections. Unfortunately, public health and patient care efforts have been hampered by the lack of FDA-cleared NAATs with claims for anorectal or oropharyngeal samples. At the time of the initiation of this study, no commercially available assays had these claims. We formed a novel partnership among academic institutions and diagnostic manufacturers to address this public health need. From May 2018 through August 2019, we recruited 1108 women, 1256 men, and 26 transgender persons each of whom provided 3 anal and 3 oropharyngeal swab specimens. The 3 anal swabs were pooled into a single transport tube as were the 3 oropharyngeal swabs. The performance of each of three study assays was estimated by comparison to the composite result and relative to one another. Percent positivity for chlamydia was 5.9 and 1.2% from anal and oropharyngeal specimens, respectively, compared to 4.2 and 4.1% for gonorrhea. Sensitivity for chlamydia detection ranged from 81.0 to 95.1% and 82.8 to 100% for anal and oropharyngeal specimens, respectively. Gonorrhea sensitivity ranged from 85.9 to 99.0% and 74.0 to 100% for anal and oropharyngeal samples, respectively. Specificity estimates were ≥ 98.9% for all assays, organisms, and sample types. Although there was heterogeneity between sensitivity estimates, these assays offer better ability to detect extragenital infections than culture and potential solutions for providing appropriate sexual health care for populations in which these infections are of concern.

Cancer is the second leading cause of mortality in EU countries, and the needs to tackle cancer are obvious. New scientific understanding, techniques and methodologies are opening up horizons for significant improvements in diagnosis and care. However, take-up is uneven, research needs and potential outstrip currently available resources, manifestly beneficial practices—such as population-level screening for lung cancer—are still not generalised, and the quality of life of patients and survivors is only beginning to be given attention it merits. This paper, mainly based on a series of multistakeholder expert workshops organised by the European Alliance for Personalised Medicine (EAPM), looks at some of those specifics in the interest of planning a way forward. Part of this exercise also involves taking account of the specific nature of Europe and its constituent countries, where the complexities of planning a way forward are redoubled by the wide variations in national and regional approaches to cancer, local epidemiology and the wide disparities in health systems. Despite all the differences between cancers and national and regional resources and approaches to cancer care, there is a common objective in pursuing broader and more equal access to the best available care for all European citizens.

ObjectivesCefiderocol is a novel siderophore cephalosporin with in vitro activity against multidrug-resistant (MDR), gram-negative bacteria and intrinsic structural stability to all classes of carbapenemases. We sought to identify gene variants that could affect the mechanism of action (MOA) of cefiderocol.MethodsWe report a case of bacteremia in a liver transplant candidate with a strain of carbapenem-resistant Escherichia coli that was found to be resistant to cefiderocol despite no prior treatment with this antimicrobial agent. Using whole-genome sequencing, we characterized the genomic content of this E coli isolate and assessed for genetic variants between related strains that were found to be cefiderocol susceptible.ResultsWe identified several variants in genes with the potential to affect the mechanism of action of cefiderocol.ConclusionsThe cefiderocol resistance in the E coli isolate identified in this study is likely due to mutations in the cirA gene, an iron transporter gene.

Cancer is the second leading cause of mortality in EU countries, and the needs to tackle cancer are obvious. New scientific understanding, techniques and methodologies are opening up horizons for significant improvements in diagnosis and care. However, take-up is uneven, research needs and potential outstrip currently available resources, manifestly beneficial practices—such as population-level screening for lung cancer—are still not generalised, and the quality of life of patients and survivors is only beginning to be given attention it merits. This paper, mainly based on a series of multistakeholder expert workshops organised by the European Alliance for Personalised Medicine (EAPM), looks at some of those specifics in the interest of planning a way forward. Part of this exercise also involves taking account of the specific nature of Europe and its constituent countries, where the complexities of planning a way forward are redoubled by the wide variations in national and regional approaches to cancer, local epidemiology and the wide disparities in health systems. Despite all the differences between cancers and national and regional resources and approaches to cancer care, there is a common objective in pursuing broader and more equal access to the best available care for all European citizens.

BackgroundGenomic profiling studies of diffuse gliomas have led to new improved classification schemes that better predict patient outcomes compared to conventional histomorphology alone. One example is the recognition that patients with IDH-wildtype diffuse astrocytic gliomas demonstrating lower-grade histologic features but genomic and/or epigenomic profile characteristic of glioblastoma typically have poor outcomes similar to patients with histologically diagnosed glioblastoma. Here we sought to determine the clinical impact of prospective genomic profiling for these IDH-wildtype diffuse astrocytic gliomas lacking high-grade histologic features but with molecular profile of glioblastoma.MethodsClinical management and outcomes were analyzed for 38 consecutive adult patients with IDH-wildtype diffuse astrocytic gliomas lacking necrosis or microvascular proliferation on histologic examination that were genomically profiled on a prospective clinical basis revealing criteria for an integrated diagnosis of "diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV" per cIMPACT-NOW criteria.ResultsWe identified that this diagnosis consists of two divergent clinical scenarios based on integration of radiologic, histologic, and genomic features that we term "early/evolving" and "undersampled" glioblastoma, IDH-wildtype. We found that prospective genomically guided identification of early/evolving and undersampled IDH-wildtype glioblastoma resulted in more aggressive patient management and improved clinical outcomes compared to a biologically matched historical control patient cohort receiving standard-of-care therapy based on histomorphologic diagnosis alone.ConclusionsThese results support routine use of genomic and/or epigenomic profiling to accurately classify glial neoplasms, as these assays not only improve diagnostic classification but critically lead to more appropriate patient management that can improve clinical outcomes.

Background: Cytopathological evaluation of thyroid fine-needle aspiration biopsy (FNAB) specimens can fail to raise preoperative suspicion of medullary thyroid carcinoma (MTC). The Afirma RNA-sequencing MTC classifier identifies MTC among FNA samples that are cytologically indeterminate, suspicious, or malignant (Bethesda categories III-VI). In this study we report the development and clinical performance of this MTC classifier. Methods: Algorithm training was performed with a set of 483 FNAB specimens (21 MTC and 462 non-MTC). A support vector machine classifier was developed using 108 differentially expressed genes, which includes the 5 genes in the prior Afirma microarray-based MTC cassette. Results: The final MTC classifier was blindly tested on 211 preoperative FNAB specimens with subsequent surgical pathology, including 21 MTC and 190 non-MTC specimens from benign and malignant thyroid nodules independent from those used in training. The classifier had 100% sensitivity (21/21 MTC FNAB specimens correctly called positive; 95% confidence interval [CI] = 83.9-100%) and 100% specificity (190/190 non-MTC FNAs correctly called negative; CI = 98.1-100%). All positive samples had pathological confirmation of MTC, while all negative samples were negative for MTC on surgical pathology. Conclusions: The RNA-sequencing MTC classifier accurately identified MTC from preoperative thyroid nodule FNAB specimens in an independent validation cohort. This identification may facilitate an MTC-specific preoperative evaluation and resulting treatment.

Not all patients produce sputum, yet most available TB tests use sputum. We investigated the utility of a novel RNA-based quantitative test, the tuberculosis molecular bacterial load assay (TB-MBLA), for the detection and quantification of Mycobacterium tuberculosis in stool. Stools from 100 adult individuals were treated with OMNIgene-sputum reagent and tested using Xpert MTB/RIF ultra (Xpert ultra), auramine O smear microscopy (smear), mycobacterial growth indicator tube (MGIT), and Lowenstein-Jensen (LJ) cultures. The remaining portions were frozen at -20°C and later tested by TB-MBLA. MGIT sputum culture was used as a TB confirmatory test and reference for stool tests. Sixty-one of 100 participants were already confirmed TB positive by MGIT sputum culture, 20 (33%) of whom were HIV coinfected. TB-MBLA detected M. tuberculosis in 57/100 stool samples, including 49 already confirmed for TB. The mean bacterial load measured by stool TB-MBLA was 5.67 ± 1.7 log10 estimated CFU (eCFU) per mL in HIV-coinfected participants, which was higher than the 4.83 ± 1.59 log10 eCFU per mL among the HIV-negative participants (P = 0.04). The sensitivities (95% confidence intervals [CI]) of stool assays were 80% (68 to 89) and 90% (79 to 98) for TB-MBLA and Xpert ultra, which were both higher than the 44% (32 to 58), 64% (51 to 76), and 62% (45 to 77) for smear, MGIT, and Lowenstein-Jensen (LJ) stool cultures, respectively. The specificity (95% CI) of stool assays was highest for smear, at 97% (87 to 100), followed by Xpert ultra at 91% (76 to 98), TB-MBLA at 79% (63 to 90), LJ at 80% (64 to 91), and MGIT at 62% (45 to 77). Twenty-six percent of MGIT and 21% of LJ stool cultures were indeterminate due to contamination. Detection and quantification of viable M. tuberculosis bacilli in stool raises its utility as an alternative to sputum as a sample type for TB diagnosis. IMPORTANCE This paper highlights the value of stool as a sample type for diagnosis of tuberculosis. While other s

Innovations in infectious disease testing have improved our abilities to detect and understand the microbial world. The 2019 novel coronavirus infectious disease (COVID-19) pandemic introduced new innovations including non-prescription "over the counter" infectious disease tests, mass spectrometry-based detection of COVID-19 host response, and the implementation of artificial intelligence (AI) and machine learning (ML) to identify individuals infected by the severe acute respiratory syndrome - coronavirus - 2 (SARS-CoV-2). As the world recovers from the COVID-19 pandemic; these innovative solutions will give rise to a new era of infectious disease tests extending beyond the detection of SARS-CoV-2. To this end, the purpose of this review is to summarize current trends in infectious disease testing and discuss innovative applications specifically in the areas of POC testing, MS, molecular diagnostics, sample types, and AI/ML.

Mass spectrometry imaging is a technique that measures the composition of tissue slices and can visualise the distribution of different components from tissue. This way, for example, the distribution of drugs can be shown or a distinction can be made between healthy and diseased tissue. This thesis aimed towards getting the imaging procedure within a clinical timeframe through the development of a new sample preparation device, where the whole workflow was reduced from 90 minutes to less than half an hour. Furthermore, it describes a new method to quantify drugs in tissue, which aids in visualizing how drugs are absorbed into tissue. Finally, the technique was used to study biopsies of cholangiocarcinoma patients. Here, a link was found between a high level of unsaturated versus saturated sulfatides (a specific type of lipids) and a poorer survival outcome.

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

Not all patients produce sputum, yet most available TB tests use sputum. We investigated the utility of a novel RNA-based quantitative test, the tuberculosis molecular bacterial load assay (TB-MBLA), for the detection and quantification of Mycobacterium tuberculosis in stool. Stools from 100 adult individuals were treated with OMNIgene-sputum reagent and tested using Xpert MTB/RIF ultra (Xpert ultra), auramine O smear microscopy (smear), mycobacterial growth indicator tube (MGIT), and Lowenstein-Jensen (LJ) cultures. The remaining portions were frozen at -20°C and later tested by TB-MBLA. MGIT sputum culture was used as a TB confirmatory test and reference for stool tests. Sixty-one of 100 participants were already confirmed TB positive by MGIT sputum culture, 20 (33%) of whom were HIV coinfected. TB-MBLA detected M. tuberculosis in 57/100 stool samples, including 49 already confirmed for TB. The mean bacterial load measured by stool TB-MBLA was 5.67 ± 1.7 log10 estimated CFU (eCFU) per mL in HIV-coinfected participants, which was higher than the 4.83 ± 1.59 log10 eCFU per mL among the HIV-negative participants (P = 0.04). The sensitivities (95% confidence intervals [CI]) of stool assays were 80% (68 to 89) and 90% (79 to 98) for TB-MBLA and Xpert ultra, which were both higher than the 44% (32 to 58), 64% (51 to 76), and 62% (45 to 77) for smear, MGIT, and Lowenstein-Jensen (LJ) stool cultures, respectively. The specificity (95% CI) of stool assays was highest for smear, at 97% (87 to 100), followed by Xpert ultra at 91% (76 to 98), TB-MBLA at 79% (63 to 90), LJ at 80% (64 to 91), and MGIT at 62% (45 to 77). Twenty-six percent of MGIT and 21% of LJ stool cultures were indeterminate due to contamination. Detection and quantification of viable M. tuberculosis bacilli in stool raises its utility as an alternative to sputum as a sample type for TB diagnosis. IMPORTANCE This paper highlights the value of stool as a sample type for diagnosis of tuberculosis. While other s

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

[EN] Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically ¿lossy¿ liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.

[EN] Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically ¿lossy¿ liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.

Mass spectrometry imaging is a technique that measures the composition of tissue slices and can visualise the distribution of different components from tissue. This way, for example, the distribution of drugs can be shown or a distinction can be made between healthy and diseased tissue. This thesis aimed towards getting the imaging procedure within a clinical timeframe through the development of a new sample preparation device, where the whole workflow was reduced from 90 minutes to less than half an hour. Furthermore, it describes a new method to quantify drugs in tissue, which aids in visualizing how drugs are absorbed into tissue. Finally, the technique was used to study biopsies of cholangiocarcinoma patients. Here, a link was found between a high level of unsaturated versus saturated sulfatides (a specific type of lipids) and a poorer survival outcome.

RNA-basierte Tools (u.a. synthetische sRNAs) wurden mit dem Ziel entworfen und entwickelt, neuartige molekulare Schaltkreise sowie therapeutische und diagnostische Ansätze für Nonsense-Mutations-assoziierte Krankheiten, insbesondere Mukoviszidose bereitzustellen. Dabei wurden zelluläres Computing zusammen mit de-novo-Ansätzen zur Genregulation in Hinblick auf Diagnose und Behandlung von Mukoviszidose verfolgt. Die vorgeschlagenen Diagnose-Regeln basieren auf Boolescher Logik und werden mittels miRNAs ausgeführt., RNA-based tools (e.g. synthetic small RNAs, sRNAs) are designed and developed for engineering novel molecular circuits and theranostic approaches of genetic nonsense mutation- associated diseases, particularly cystic fibrosis (CF). Cellular computation combined with de novo approaches to gene regulation were explored for the diagnosis and mitigation of CF. Diagnostic rules based on Boolean expressions using miRNA profiles were proposed., CONACYT-DAAD 2017 Fellowship

Innovations in infectious disease testing have improved our abilities to detect and understand the microbial world. The 2019 novel coronavirus infectious disease (COVID-19) pandemic introduced new innovations including non-prescription "over the counter" infectious disease tests, mass spectrometry-based detection of COVID-19 host response, and the implementation of artificial intelligence (AI) and machine learning (ML) to identify individuals infected by the severe acute respiratory syndrome - coronavirus - 2 (SARS-CoV-2). As the world recovers from the COVID-19 pandemic; these innovative solutions will give rise to a new era of infectious disease tests extending beyond the detection of SARS-CoV-2. To this end, the purpose of this review is to summarize current trends in infectious disease testing and discuss innovative applications specifically in the areas of POC testing, MS, molecular diagnostics, sample types, and AI/ML.

Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location.

Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location.

The qualitative ID Now COVID-19 assay combines claimed performance and ease of use that seem to position it as a reliable test for urgent patient management. However, the declared limit of detection (LOD) of 125 genome equivalents/mL is not confirmed by the published studies, which observed a range of LOD varying from 276 to 20.000 copies/mL. We decided to establish the LOD value on more robust basis using serial dilutions of a SARS-CoV-2 culture supernatant sample of defined concentration. Afterwards, we tested the analytical performances of the assay with 23 QCMD external quality control measurements. Hence, taking into consideration the additional dilution in the sample receiver cup, we found a lower 95 % LOD of 64 copies/mL. For its intended use and with the new established LOD, ID Now COVID-19 proved to be a suitable test for the diagnosis of COVID-19 in contagious patients, as proposed by the latest Belgian recommendations.

Optofluidics, the science that integrates photonics and microfluidics, has produced a number of promising devices for biosensing and bioanalysis. In the past couple of years, several optofluidic approaches have demonstrated the capability to optically detect single biological microparticles. However, despite the sensitivity, many other requirements must be met to fully realize a molecular diagnostic device on a lab-on-a-chip platform. These requirements include target specificity, large dynamic range of detection, a low limit of detection, and multiplex differentiated diagnosis of many relevant biomarkers in a single sample. To this end, we have previously developed an optofluidic biosensor based on liquid-core antiresonant reflecting optical waveguides (LC-ARROWs). Orthogonally intersecting liquid and solid core ARROWs deliver the appropriate architecture for a highly sensitive, reconfigurable, and portable to the point-of-care device for ultra-sensitive detection of fluorescently labeled biomarkers in flow. We have recently introduced multiplexing capabilities into the ARROW-based optofluidic platform by integrating multi-mode interference (MMI) excitation waveguides, allowing for spectral and spatial multiplexing on a single compact chip. As a proof of concept, influenza virions were non-specifically labeled and simultaneously and differentially detected on chip. The goal of this thesis was to expand the detection capabilities of the MMI waveguide-detection platform via developing different specific bioassays to analyze different molecular biomarkers on chip and in clinically relevant environments and concentrations. First, a bead-based nucleic acid capture assay is described, which specifically captures nucleic acids onto an immobilization magnetic microsphere and functionalizes the complex with fluorescent dyes. The first simultaneous, multiplex differentiated detection of non-amplified total RNA samples from Viral Hemorrhagic Fever (VHF) infected cell cultures

The following is a concise review of the Pediatric Pulmonary Medicine Core reviewing pediatric pulmonary infections, diagnostic assays, and imaging techniques presented at the 2021 American Thoracic Society Core Curriculum. Molecular methods have revolutionized microbiology. We highlight the need to collect appropriate samples for detection of specific pathogens or for panels and understand the limitations of the assays. Considerable progress has been made in imaging modalities for detecting pediatric pulmonary infections. Specifically, lung ultrasound and lung magnetic resonance imaging are promising radiation-free diagnostic tools, with results comparable with their radiation-exposing counterparts, for the evaluation and management of pulmonary infections. Clinicians caring for children with pulmonary disease should ensure that patients at risk for nontuberculous mycobacteria disease are identified and receive appropriate nontuberculous mycobacteria screening, monitoring, and treatment. Children with coronavirus disease (COVID-19) typically present with mild symptoms, but some may develop severe disease. Treatment is mainly supportive care, and most patients make a full recovery. Anticipatory guidance and appropriate counseling from pediatricians on social distancing and diagnostic testing remain vital to curbing the pandemic. The pediatric immunocompromised patient is at risk for invasive and opportunistic pulmonary infections. Prompt recognition of predisposing risk factors, combined with knowledge of clinical characteristics of microbial pathogens, can assist in the diagnosis and treatment of specific bacterial, viral, or fungal diseases.

BackgroundAirway infections are difficult to distinguish from acute rejection in lung transplant recipients. Traditional culture techniques take time that may delay treatment. We hypothesized that a rapid multiplex molecular assay could improve time to diagnosis and appropriate clinical decision making.MethodsIn a prospective observational study of recipients undergoing bronchoscopy, we assessed the BioFire® FilmArray® Pneumonia Panel (BFPP) in parallel to standard of care (SOC) diagnostics. Research clinicians performed shadow (research only) clinical decision making in real time. Time to report and interpretation were reported as median and interquartile ranges and compared by Wilcoxon signed-ranked test. Agreement was defined based on detection of any species targeted in the molecular assay.ResultsFor the 150 enrolled subjects, BFPP results were available 3.8 hours (IQR 2.8-5.1) following bronchoscopy, compared to 13 hours for viral SOC (IQR 10-34, P < .001) results and 48 hours for bacterial SOC (IQR 46-70, P < .001) results. Positive BFPP results were interpreted in 9 hours (IQR 5-20) following bronchoscopy, compared to 74 hours for SOC (IQR 37-110, P < .001). Assays agreed for 138 (92%) of the 150 subjects. Of 22 BFPP diagnoses, five (23%) resulted in a shadow antibiotic recommendation. Notable BFPP deficiencies included fungal species and H parainfluenzae, accounting for 15 (27%) and 13 (23%) of the 56 actionable SOC results, respectively.ConclusionsThis molecular diagnostic including bacterial targets has the potential to shorten time to diagnosis and augment current clinical decision making but cannot replace SOC culture methods.

Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location.

The following is a concise review of the Pediatric Pulmonary Medicine Core reviewing pediatric pulmonary infections, diagnostic assays, and imaging techniques presented at the 2021 American Thoracic Society Core Curriculum. Molecular methods have revolutionized microbiology. We highlight the need to collect appropriate samples for detection of specific pathogens or for panels and understand the limitations of the assays. Considerable progress has been made in imaging modalities for detecting pediatric pulmonary infections. Specifically, lung ultrasound and lung magnetic resonance imaging are promising radiation-free diagnostic tools, with results comparable with their radiation-exposing counterparts, for the evaluation and management of pulmonary infections. Clinicians caring for children with pulmonary disease should ensure that patients at risk for nontuberculous mycobacteria disease are identified and receive appropriate nontuberculous mycobacteria screening, monitoring, and treatment. Children with coronavirus disease (COVID-19) typically present with mild symptoms, but some may develop severe disease. Treatment is mainly supportive care, and most patients make a full recovery. Anticipatory guidance and appropriate counseling from pediatricians on social distancing and diagnostic testing remain vital to curbing the pandemic. The pediatric immunocompromised patient is at risk for invasive and opportunistic pulmonary infections. Prompt recognition of predisposing risk factors, combined with knowledge of clinical characteristics of microbial pathogens, can assist in the diagnosis and treatment of specific bacterial, viral, or fungal diseases.

[Simple Summary] A solitary fibrous tumor (SFT) is a rare mesenchymal neoplasm that can arise at any body location. Local or distant recurrences occur in a significant proportion of cases, but these recurrences are difficult to predict using clinical or pathological features. A specific genetic alteration, the gene fusion NAB2-STAT6, is considered to be the defining driver mutation, and different fusion variants seem to account for specific clinical and pathological features, but their prognostic value remains controversial. We inspected a series of 83 SFTs with a high rate of recurrence to evaluate the clinical significance of several potential biomarkers in addition to gene fusion. Our findings confirm previous observations and uncover novel associations of molecular alterations with clinical features, adding additional evidence for their potential application as molecular biomarkers that are helpful to predict the course of the disease., [Abstract] Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location.

Aims: The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. Methods and Results: The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter technology, FoundationOne and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. Conclusion(s): Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease.Copyright © 2020 The Authors. Histopathology published by John Wiley & Sons Ltd

Aims: The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. Methods and Results: The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter technology, FoundationOne and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. Conclusion(s): Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease.Copyright © 2020 The Authors. Histopathology published by John Wiley & Sons Ltd

This study evaluated and compared the accuracy of BCID2 with that of BCID to identify bacterial species and relative antimicrobial resistance genes directly from positive BCs. We used archived samples from positive BCs (1.5 ml thereof mixed with 100 ml of dimethyl sulfoxide and stored at 280°C), which had prospectively been processed with the BD Bactec 9240 (BD Diagnostic Systems, Sparks, MD), BacTAlert 3D (bioMérieux), or BacTAlert Virtuo (bioMérieux) system at two hospital microbiology laboratories from January 2018 to August 2020.

Optofluidics, the science that integrates photonics and microfluidics, has produced a number of promising devices for biosensing and bioanalysis. In the past couple of years, several optofluidic approaches have demonstrated the capability to optically detect single biological microparticles. However, despite the sensitivity, many other requirements must be met to fully realize a molecular diagnostic device on a lab-on-a-chip platform. These requirements include target specificity, large dynamic range of detection, a low limit of detection, and multiplex differentiated diagnosis of many relevant biomarkers in a single sample. To this end, we have previously developed an optofluidic biosensor based on liquid-core antiresonant reflecting optical waveguides (LC-ARROWs). Orthogonally intersecting liquid and solid core ARROWs deliver the appropriate architecture for a highly sensitive, reconfigurable, and portable to the point-of-care device for ultra-sensitive detection of fluorescently labeled biomarkers in flow. We have recently introduced multiplexing capabilities into the ARROW-based optofluidic platform by integrating multi-mode interference (MMI) excitation waveguides, allowing for spectral and spatial multiplexing on a single compact chip. As a proof of concept, influenza virions were non-specifically labeled and simultaneously and differentially detected on chip. The goal of this thesis was to expand the detection capabilities of the MMI waveguide-detection platform via developing different specific bioassays to analyze different molecular biomarkers on chip and in clinically relevant environments and concentrations. First, a bead-based nucleic acid capture assay is described, which specifically captures nucleic acids onto an immobilization magnetic microsphere and functionalizes the complex with fluorescent dyes. The first simultaneous, multiplex differentiated detection of non-amplified total RNA samples from Viral Hemorrhagic Fever (VHF) infected cell cultures

BackgroundAirway infections are difficult to distinguish from acute rejection in lung transplant recipients. Traditional culture techniques take time that may delay treatment. We hypothesized that a rapid multiplex molecular assay could improve time to diagnosis and appropriate clinical decision making.MethodsIn a prospective observational study of recipients undergoing bronchoscopy, we assessed the BioFire® FilmArray® Pneumonia Panel (BFPP) in parallel to standard of care (SOC) diagnostics. Research clinicians performed shadow (research only) clinical decision making in real time. Time to report and interpretation were reported as median and interquartile ranges and compared by Wilcoxon signed-ranked test. Agreement was defined based on detection of any species targeted in the molecular assay.ResultsFor the 150 enrolled subjects, BFPP results were available 3.8 hours (IQR 2.8-5.1) following bronchoscopy, compared to 13 hours for viral SOC (IQR 10-34, P < .001) results and 48 hours for bacterial SOC (IQR 46-70, P < .001) results. Positive BFPP results were interpreted in 9 hours (IQR 5-20) following bronchoscopy, compared to 74 hours for SOC (IQR 37-110, P < .001). Assays agreed for 138 (92%) of the 150 subjects. Of 22 BFPP diagnoses, five (23%) resulted in a shadow antibiotic recommendation. Notable BFPP deficiencies included fungal species and H parainfluenzae, accounting for 15 (27%) and 13 (23%) of the 56 actionable SOC results, respectively.ConclusionsThis molecular diagnostic including bacterial targets has the potential to shorten time to diagnosis and augment current clinical decision making but cannot replace SOC culture methods.

In this work, we present the design and fabrication of a fiber device that performs digital droplet microfluidics for molecular diagnostics. A variety of fibers and capillaries were used to build three connected modules dedicated to droplet generation, incubation, and fluorescence detection which enables a uniaxial arrangement. This is in contrast to the traditional 2-dimensional lab-on-a-chip architecture. We characterize our fiber device using a fluorescein dilution series. Our observed detection limit is on the order of 10 nM fluorescein. We demonstrate our all-fiber device for the fluorescence readout after loop-mediated isothermal amplification (LAMP) of synthetic SARS-CoV-2. Our results suggest that this fiber device can successfully distinguish between positive and negative samples in molecular diagnostics. We propose that our fiber device offers benefits over microfluidic chip techniques such as easier optical integration, much simpler sample loading, and faster diagnosis with high specificity and sensitivity., Part of proceedings, ISBN 978-1-5106-4381-9, QC 20230117

The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

We assessed the real-world utility of universal broad-range polymerase chain reaction sequencing for pathogen detection. Among 1062 clinical samples, 107/1062 (10.1%) had a clinically significant, positive result, with substantial variation by specimen type. Clinical management was changed in 44/1062 (4.1%). These data can help maximize utility of this emerging diagnostic.