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1.
Discov Med ; 29(157): 129-137, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002409

RESUMO

Sepsis is a life-threatening clinical condition demanding accurate and rapid diagnosis of the culprit pathogen, thereby to improve prognosis. Pathogen determination through blood culture is the gold standard for diagnosis but has limitations due to low sensitivity. Recently, circulating DNAs derived from pathogenic organisms were found in the plasma of patients with sepsis and were further proved to be more sensitive biomarkers for the diagnosis of the pathogen origin in sepsis. However, the fundamental molecular characteristics of circulating DNA in patients with sepsis remain unclear. Here, we used specific PCR and Sanger sequencing to verify the microbiology culture results via the corresponding plasma circulating DNA. We analyzed the composition and molecular characteristics of circulating DNA in septic patients using next-generation sequencing technology. We showed the presence of pathogen-derived circulating DNA in the plasma of patients with sepsis. The sizes of circulating DNA fragments derived from pathogenic bacteria showed a skewed unimodal distribution, while those derived from host cells showed a normal unimodal distribution. Lengths of fragments at peak concentration for both origins ranged from 150 bp to 200 bp, and reads mapping to pathogenic bacteria genome distributed uniformly on the reference. Our findings have improved our understanding of microbial circulating DNA in patients with sepsis as a potential methodology for the accurate diagnosis of sepsis, especially in light of an urgent need for such a diagnosis associated with the COVID-19 infection.


Assuntos
Infecções Bacterianas/microbiologia , Ácidos Nucleicos Livres/sangue , DNA Bacteriano/sangue , Sepse/microbiologia , Adulto , Idoso , Infecções Bacterianas/complicações , Infecções Bacterianas/diagnóstico , Betacoronavirus , Ácidos Nucleicos Livres/análise , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Técnicas de Cultura , DNA Bacteriano/análise , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Pandemias , Pneumonia Viral , Reação em Cadeia da Polimerase , Sepse/complicações , Sepse/diagnóstico , Análise de Sequência de DNA
2.
PLoS One ; 15(8): e0238119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845896

RESUMO

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of ≤16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.


Assuntos
Líquido Ascítico/química , Ácidos Nucleicos Livres/análise , DNA Bacteriano/análise , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Abdome/microbiologia , Abdome/patologia , Adolescente , Adulto , Idoso , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Tuberculose/patologia , Adulto Jovem
3.
PLoS One ; 15(8): e0238245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845907

RESUMO

To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors.


Assuntos
Mapeamento Cromossômico/métodos , Limite de Detecção , Teste Pré-Natal não Invasivo/métodos , Sequenciamento Completo do Genoma/métodos , Ácidos Nucleicos Livres/análise , Deleção Cromossômica , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 1/genética , Síndrome do Miado do Gato/diagnóstico , Síndrome do Miado do Gato/genética , Síndrome de DiGeorge/diagnóstico , Síndrome de DiGeorge/genética , Feminino , Humanos , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Gravidez , Cuidado Pré-Natal , Síndrome de Wolf-Hirschhorn/diagnóstico , Síndrome de Wolf-Hirschhorn/genética
4.
Transplant Proc ; 52(9): 2592-2595, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32800517

RESUMO

BACKGROUND: Kidney allograft biopsy is the gold standard for diagnosis of rejection. Under the current extraordinary circumstances of the coronavirus disease 2019 (COVID-19), in which social distancing is key to limiting the spread of the virus, the model used to provide care to transplant recipients has undergone a very rapid transformation. In the spirit of medical distancing, we have been using the donor-derived cell-free DNA (dd-cfDNA) test for screening for rejection. METHODS: This article describes our experience with this approach between March 15th and May 20th, 2020. RESULTS: This test was obtained for-cause in 23 patients and for monitoring in 9 patients. Normal results aided in forgoing biopsy in 63% of the patients for whom the test was obtained in the outpatient setting. The test is neither 100% sensitive nor specific for rejection; however, when used in combination with the available clinical information, it can be used for determining whether bringing in a transplant recipient into a medical facility is necessary. CONCLUSIONS: In the event COVID-19 becomes a long-term challenge for our community, noninvasive biomarkers such as the dd-cfDNA may become more relevant than ever in enhancing our ability to care for our transplant patients while maximizing the distancing measures.


Assuntos
Ácidos Nucleicos Livres/análise , Infecções por Coronavirus/prevenção & controle , Transmissão de Doença Infecciosa/prevenção & controle , Rejeição de Enxerto/diagnóstico , Transplante de Rim/efeitos adversos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Adulto , Aloenxertos/química , Betacoronavirus , Biomarcadores/análise , Infecções por Coronavirus/transmissão , Feminino , Humanos , Rim/química , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/transmissão , Transplante Homólogo
5.
Med Hypotheses ; 142: 109812, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32416412

RESUMO

The fast-growing outbreak of 2019 novel coronaviruses (SARS-CoV-2) reached all continents except the Antarctica in merely three months. Severe SARS-CoV-2 infection (COVID-19) has a bad clinical outcome, and some reports emphasized the role of cytokine storm and dysfunctions of multiple organs. However, the etiology of severe COVID-19 has been largely unknown. Similar as SARS-CoV and MERS-CoV, SARS-CoV-2 is also thought derived from bat coronaviruses. However, it is not pathogenic for bat at all, because free DNA in cytoplasm or blood cannot bring up violent immune response in bat; but it can produce severe inflammations in human. I hypothesized that the damage induced by free DNA is a reason for severe COVID-19, which can explain many symptoms of this disease, such as cytokine storm, acute respiratory distress syndrome (ARDS) and muscus plug, acute injuries of heart, liver and kidney, and some special symptoms of COVID-19. My hypothesis will be helpful for better understand the etiology of severe COVID-19.


Assuntos
Ácidos Nucleicos Livres/análise , Infecções por Coronavirus/sangue , Infecções por Coronavirus/metabolismo , DNA Viral/análise , Pneumonia Viral/sangue , Pneumonia Viral/metabolismo , Animais , Betacoronavirus , Quirópteros , Doenças Transmissíveis , Infecções por Coronavirus/transmissão , Citocinas/metabolismo , Citoplasma/virologia , Surtos de Doenças , Humanos , Inflamação , Modelos Teóricos , Pandemias , Pneumonia Viral/transmissão
6.
Neoplasma ; 67(4): 909-915, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32386482

RESUMO

Hepatocellular carcinoma (HCC) is the third deadliest cancer in the world with high morbidity and poor prognosis. CTCFL (CCCTC-binding factor like) is a member of the cancer testis antigen (CTA) family with oncogenic properties. To demonstrate whether the hypomethylation of CTCFL promoters in plasma could be used as a noninvasive biomarker to predict poor prognosis of HCC, we extracted cell-free DNA from the plasma and detected the methylation status of CTCFL in 43 HCC, 5 liver cirrhosis and 6 benign lesion samples using methylation specific PCR (MSP). Our study indicated that the hypomethylation of CTCFL promoters in HCC plasma samples (60.4%) was significantly different from that in benign lesion plasma samples (16.7%) with a p-value of 0.043. Analysis of clinicopathological data showed that the methylation status of CTCFL promoters was significantly correlated with microvascular involvement (MVI) (p=0.001) and postoperative recurrence (p=0.031). Furthermore, clinical prognosis data of 347 HCC patients from The Cancer Genome Atlas (TCGA) database displayed that the hypomethylated group had worse overall survival than the hypermethylated group (p=0.0056). In conclusion, we provide evidence that the hypomethylation of CTCFL promoters in cell-free DNA is a biomarker for monitoring HCC patients, which can be used as a noninvasive prediction index for tumor recurrence and provide the individualized decision-making for clinicians.


Assuntos
Carcinoma Hepatocelular , Proteínas de Ligação a DNA , Neoplasias Hepáticas , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Ácidos Nucleicos Livres/análise , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Recidiva Local de Neoplasia/genética , Prognóstico , Regiões Promotoras Genéticas
7.
Cancer Cytopathol ; 128(8): 545-552, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32286726

RESUMO

BACKGROUND: Epidermal growth factor receptor (EGFR) is an important marker for targeted therapy in patients with advanced non-small cell lung cancer (NSCLC). The samples obtained with minimally invasive biopsy techniques are usually small, and this limits their application in tissue subtyping or molecular profiling. The supernatants obtained after centrifugation of fine-needle aspiration (FNA) samples are typically discarded. However, these fractions might contain cell-free DNA that could be tested for EGFR mutations by genotyping methods that are normally used for plasma analysis. METHODS: In this study, 214 patients with known or suspected NSCLC who underwent FNA were enrolled. The workflow of the supernatants before molecular detection was as follows. The discarded FNA samples (15 mL) were stored in CytoLyt, a cleaning, fixation solution, and 10 mL of each sample was placed in a preservation solution for separation by low-speed centrifugation. The primary supernatants (8 mL) were then separated by high-speed centrifugation to obtain secondary supernatants. DNA was extracted from the supernatants with QIAamp circulating nucleic acid kits (Qiagen) and circulating DNA kits (AmoyDx), and EGFR mutations were assessed with Super-ARMS EGFR detection kits (AmoyDx). The DNA was then extracted from corresponding cell pellets with tissue DNA kits (AmoyDx), and the EGFR status was analyzed with the amplification refractory mutation system and next-generation sequencing methods. RESULTS: All 214 samples yielded an adequate amount of cell-free DNA for EGFR detection. The use of different DNA commercial extraction kits and the DNA contents of tumor cells did not affect the yield of DNA from the supernatants. The external controlled cycle threshold value of the EGFR test was affected by the concentration of the DNA in the supernatants (P < .05). However, the difference in the concentrations of the DNA in the supernatants did not affect the EGFR mutation status. The EGFR-positive rate was 57.5% (123 of 214) in both the supernatants and the pellets from the 214 FNA samples. The concordance between EGFR variants in the supernatants and the corresponding pellets was 97.2%. EGFR mutations were also detected in 3 pellets but not in their corresponding supernatants and in 3 supernatants but not in their corresponding pellets. The supernatants of FNA biopsy samples might represent a new source for gaining information regarding the molecular characteristics of patients for targeted therapy. CONCLUSIONS: Discarded supernatants provided an adequate amount of cell-free DNA for EGFR detection, and this means that the pellets can be reserved for additional morphological and molecular analyses or to avoid repeat biopsies. Analyzing the EGFR status in cell supernatants and pellets might improve detection sensitivity and confer benefits to patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Ácidos Nucleicos Livres/análise , Neoplasias Pulmonares/genética , Mutação , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Receptores ErbB/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade
9.
Sci Rep ; 10(1): 6083, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269293

RESUMO

PURPOSE: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. EXPERIMENTAL DESIGN: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. RESULTS: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = <0.001). Similarly, circulating tumoral HPV-RNA was detected at a higher sensitivity in cf-RNA compared to EV-RNA, at 83% vs. 50% (p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. CONCLUSION: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.


Assuntos
Carcinoma de Células Escamosas/patologia , Ácidos Nucleicos Livres/análise , DNA Viral/análise , Vesículas Extracelulares/virologia , Testes de DNA para Papilomavírus Humano/métodos , Neoplasias Orofaríngeas/patologia , Idoso , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/virologia , Ácidos Nucleicos Livres/genética , DNA Viral/genética , Feminino , Testes de DNA para Papilomavírus Humano/normas , Humanos , Limite de Detecção , Biópsia Líquida/métodos , Biópsia Líquida/normas , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/sangue , Neoplasias Orofaríngeas/virologia
10.
Microbiome ; 8(1): 18, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046792

RESUMO

BACKGROUND: Cell-free DNA (cfDNA) in blood, urine, and other biofluids provides a unique window into human health. A proportion of cfDNA is derived from bacteria and viruses, creating opportunities for the diagnosis of infection via metagenomic sequencing. The total biomass of microbial-derived cfDNA in clinical isolates is low, which makes metagenomic cfDNA sequencing susceptible to contamination and alignment noise. RESULTS: Here, we report low biomass background correction (LBBC), a bioinformatics noise filtering tool informed by the uniformity of the coverage of microbial genomes and the batch variation in the absolute abundance of microbial cfDNA. We demonstrate that LBBC leads to a dramatic reduction in false positive rate while minimally affecting the true positive rate for a cfDNA test to screen for urinary tract infection. We next performed high-throughput sequencing of cfDNA in amniotic fluid collected from term uncomplicated pregnancies or those complicated with clinical chorioamnionitis with and without intra-amniotic infection. CONCLUSIONS: The data provide unique insight into the properties of fetal and maternal cfDNA in amniotic fluid, demonstrate the utility of cfDNA to screen for intra-amniotic infection, support the view that the amniotic fluid is sterile during normal pregnancy, and reveal cases of intra-amniotic inflammation without infection at term. Video abstract.


Assuntos
Ácidos Nucleicos Livres/análise , Biologia Computacional/métodos , DNA Bacteriano/análise , Metagenoma , Análise de Sequência de DNA/métodos , Líquido Amniótico/microbiologia , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/urina , Corioamnionite/microbiologia , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/urina , Estudos Transversais , Análise de Dados , Reações Falso-Positivas , Feminino , Feto/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Inflamação , Masculino , Gravidez , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/microbiologia , Software
11.
Prog. obstet. ginecol. (Ed. impr.) ; 63(1): 6-9, ene.-feb. 2020. graf
Artigo em Espanhol | IBECS | ID: ibc-197726

RESUMO

OBJETIVO: evaluar la disminución de la tasa de técnicas invasivas de diagnóstico prenatal tras la introducción del cribado contingente de cromosomopatías con test de DNA fetal libre circulante (DNA-lc). MATERIAL Y MÉTODOS: estudio observacional prospectivo y estudio de coste efectividad. Se describen los resultados del cribado combinado en dos tiempos de primer trimestre desde febrero de 2008 a junio de 2018: 21.744 cribados realizados de un total de 23.000 partos. Se implementa en abril de 2016 el cribado contingente de cromosomopatías con test de DNA-lc y se analizan los resultados obtenidos hasta marzo de 2019. RESULTADOS: se observa una disminución total de las técnicas invasivas del 54% a expensas de una disminución de la tasa de amniocentesis, manteniéndose constante la tasa de biopsias coriales. Se han evaluado seis indicadores de calidad. Se ha conseguido un ahorro de 70.200 euros con la implementación del test de DNA-lc. CONCLUSIONES: el test de DNA-lc resulta muy útil en el cribado contingente de cromosomopatías porque reduce la tasa de amniocentesis por indicación de alto riesgo y además es coste efectivo


OBJECTIVE: To evaluate the decrease in the rate of invasive techniques of prenatal diagnosis after the introduction of contingent screening of aneuploidies with circulating fetal free DNA test (DNA-lc). MATERIAL AND METHODS: Prospective observational study and cost effectiveness study. The results of the combined screening from February 2008 to June 2018 are described: 21.744 screenings performed of a total of 23,000 deliveries. In April 2016 the contingent screening of aneuploidies with DNA-lc test was implemented and the results until March 2019 are analyzed. RESULTS: It was observed a decrease in invasive techniques of 54% with a decrease in the amniocentesis rate, keeping the rate of corial biopsies constant. Six quality indicators have been evaluated. 70,200 euros have been achieved with the implementation of the DNA-lc test. CONCLUSIONS: The DNA-lc test is very useful in the contingent screening of aneuploidies because it decreases the amniocentesis rate by indicating high risk and it is also cost-effective


Assuntos
Humanos , Feminino , Gravidez , Ácidos Nucleicos Livres/análise , Aneuploidia , Amniocentese , Testes Genéticos/métodos , Testes para Triagem do Soro Materno/métodos , Ácidos Nucleicos Livres/sangue , Testes Genéticos/economia , Testes para Triagem do Soro Materno/economia , Estudos Prospectivos , Análise Custo-Benefício , Biópsia , Hospitais Universitários , Espanha
12.
J Clin Endocrinol Metab ; 105(3)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913467

RESUMO

CONTEXT: There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death. OBJECTIVE: To assess the performance of three unmethylated insulin DNA assays. DESIGN AND PARTICIPANTS: Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay. RESULTS: All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays. CONCLUSIONS: The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful.


Assuntos
Biomarcadores/sangue , Morte Celular , Ácidos Nucleicos Livres/sangue , Células Secretoras de Insulina/fisiologia , Insulina/genética , Adulto , Idoso , Bioensaio/normas , Biomarcadores/análise , Morte Celular/genética , Ácidos Nucleicos Livres/análise , Metilação de DNA , Feminino , Humanos , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Ensaio de Proficiência Laboratorial , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
13.
Dig Dis Sci ; 65(8): 2294-2301, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31925676

RESUMO

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.


Assuntos
Ácidos Nucleicos Livres/análise , Cisto Pancreático/química , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Cromograninas/genética , Análise Mutacional de DNA , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Masculino , Pessoa de Meia-Idade , Cisto Pancreático/genética , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/genética , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto Jovem
14.
J Gynecol Obstet Hum Reprod ; 49(1): 101624, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31472269

RESUMO

Despite many advances in assisted reproductive technology (ART), the most viable embryo selection remains a challenge for infertility treatment. This study was designed to investigate whether intra-follicular circulating cell-free DNA (cfDNA) fragments and Melatonin levels predict embryo quality in patients undergoing IVF treatment. A total of eight hundred and ninety-five follicular fluid (ff) samples were collected from 325 infertile patients undergoing IVF treatment. Patients were enrolled from August 2017 to December 2018 in the infertility center of a tertiary care hospital. A clear non-hematic follicular fluid was aspirated after the removal of eggs from the dominant follicles (>18mm) of each patient. Melatonin and E2 levels in each follicular sample were estimated by immune-chemiluminescence using commercially available kits. ALU-qPCR evaluated cfDNA levels in individual follicular fluid samples. Our study presented a significant and negative relationship between intra-follicular cfDNA and melatonin concentration (-0.541: P<0.001). Each individual follicle contains measurable copy number of cfDNA [mean: 1.85±2.98ng/µl (median; 1.86ng/µl (95% Cl: 0.96-2.87)]. In pregnant women cfDNA copy number was significantly decreased in follicular fluid samples(ff) aspirated from matured oocytes than in immature ones [p<0.01; ß = -0.42±0.49; median; 1.45ng/ml (95% Cl: 0.36-2.97) vs. 3.57ng/µl (95% Cl: 0.37-4.01) respectively. While melatonin concentration in ff samples corresponding to mature oocytes was significantly higher than in ff samples related to immature oocytes (p<0.001). Moreover, in pregnant women cfDNA level was significantly lower in ff samples related to oocytes which produces top-quality embryos versus low quality embryos [p<0.001; ß=1.81±0.91; median; 1.25ng/µl (95% Cl: 0.35-1.97)] vs. [(median; 3.65ng/ml (95% Cl: 1.23-6.36)] respectively. Likewise, in non-pregnant women melatonin levels were significantly decreased in ff samples related to embryos with high fragmentation rate (≥25%) than embryos with low fragmentation rate (<25%; p<0.001). Conclusively, this study indicates that Intra-follicular cfDNA and melatonin concentration possibly a new supplemental tool that supports to establish an advanced non-invasive early prognostic test for the patients undergoing IVF/ICSI procedure.


Assuntos
Ácidos Nucleicos Livres/análise , DNA/análise , Embrião de Mamíferos , Fertilização In Vitro , Líquido Folicular/química , Melatonina/análise , Adulto , Gonadotropina Coriônica/sangue , Variações do Número de Cópias de DNA , Fragmentação do DNA , Estradiol/análise , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Infertilidade Feminina/sangue , Infertilidade Masculina , Masculino , Oócitos/química , Folículo Ovariano/química , Indução da Ovulação/métodos , Gravidez , Estudos Prospectivos , Curva ROC
15.
N Biotechnol ; 55: 19-29, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-31580920

RESUMO

The term 'liquid biopsy', introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes. The analysis of the liquid biopsy relies on a plethora of methods whose standardisation cannot be accomplished without disclosing criticisms related to the pre-analytical phase. Thus, pre-analytical factors potentially influencing downstream cellular and molecular analyses must be considered in order to translate the liquid biopsy approach into clinical practice. The present review summarises the most recent reports in this field, discussing the main pre-analytical aspects related to CTCs, cfNAs and exosomes in blood samples for liquid biopsy analysis. A short discussion on non-blood liquid biopsy samples is also included.


Assuntos
Biópsia Líquida/métodos , Fase Pré-Analítica/métodos , Animais , Líquidos Corporais/metabolismo , Ácidos Nucleicos Livres/análise , Exossomos/metabolismo , Humanos , Células Neoplásicas Circulantes/patologia
16.
Cancer Treat Rev ; 83: 101951, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874446

RESUMO

Molecular profiling of tumor derived cell free DNA (cfDNA) is gaining ground as a prognostic and predictive biomarker. However to what extent cfDNA reflects the full metastatic landscape as currently determined by tumor tissue analysis remains controversial. Though technically challenging, whole exome sequencing (WES) of cfDNA enables thorough evaluation of somatic alterations. Here, we review the feasibility of WES of cfDNA and determine the sensitivity of WES-detected single nucleotide variants (SNVs) in cfDNA on individual patient data level using paired tumor tissue as reference (sharedSNVsAlltissueSNVs×100%). The pooled sensitivity was 50% (95% credible interval (CI): 29-72%). The tissue mutant allele frequency (MAF) of variants exclusively identified in tissue was significantly lower (12.5%, range: 0.5-18%) than the tissue MAF of variants identified in both tissue and cfDNA (23.9%, range: 17-38%), p = 0.004. The overall agreement (sharedSNVsAllSNVs×100%)between SNVs in cfDNA and tumor tissue was 31% (95% CI: 15-49%). The number of detected SNVs was positively correlated with circulating tumor DNA (ctDNA) fraction (p = 0.016). A sub analysis of samples with ctDNA fractions ≥ 25% improved the sensitivity to 69% (95% CI: 46-89%) and agreement to 46% (95% CI: 36-59%), suggesting that WES is mainly feasible for patients with high ctDNA fractions. Pre- and post-analytical procedures were highly variable between studies rendering comparisons problematic. In conclusion, various aspects of WES of cfDNA are largely in its investigative phase, standardization of methodologies is highly needed to bring this promising technique to its clinical potential.


Assuntos
Teorema de Bayes , Biomarcadores Tumorais/análise , Ácidos Nucleicos Livres/análise , DNA Tumoral Circulante/análise , DNA de Neoplasias/análise , Neoplasias/patologia , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Neoplasias/genética , Neoplasias/terapia , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma
17.
Genet Res (Camb) ; 101: e11, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31813398

RESUMO

BACKGROUND: Non-invasive prenatal testing (NIPT) for the detection of foetal aneuploidy through analysis of cell-free DNA (cfDNA) in maternal blood is offered routinely by many healthcare providers across the developed world. This testing has recently been recommended for evaluative implementation in the UK National Health Service (NHS) foetal anomaly screening pathway as a contingent screen following an increased risk of trisomy 21, 18 or 13. In preparation for delivering a national service, we have implemented cfDNA-based NIPT in our Regional Genetics Laboratory. Here, we describe our validation and verification processes and initial experiences of the technology prior to rollout of a national screening service. METHODS: Data are presented from more than 1000 patients (215 retrospective and 840 prospective) from 'high- and low-risk pregnancies' with outcome data following birth or confirmatory invasive prenatal sampling. NIPT was by the Illumina Verifi® test. RESULTS: Our data confirm a high-fidelity service with a failure rate of ~0.24% and a high sensitivity and specificity for the detection of foetal trisomy 13, 18 and 21. Secondly, the data show that a significant proportion of patients continue their pregnancies without prenatal invasive testing or intervention after receiving a high-risk cfDNA-based result. A total of 46.5% of patients referred to date were referred for reasons other than high screen risk. Ten percent (76/840 clinical service referrals) of patients were referred with ultrasonographic finding of a foetal structural anomaly, and data analysis indicates high- and low-risk scan indications for NIPT. CONCLUSIONS: NIPT can be successfully implemented into NHS regional genetics laboratories to provide high-quality services. NHS provision of NIPT in patients with high-risk screen results will allow for a reduction of invasive testing and partially improve equality of access to cfDNA-based NIPT in the pregnant population. Patients at low risk for a classic trisomy or with other clinical indications are likely to continue to access cfDNA-based NIPT as a private test.


Assuntos
Ácidos Nucleicos Livres/análise , Testes Genéticos/métodos , Teste Pré-Natal não Invasivo/métodos , Aneuploidia , Ácidos Nucleicos Livres/genética , Síndrome de Down/genética , Feminino , Feto , Humanos , Masculino , Programas Nacionais de Saúde , Gravidez , Diagnóstico Pré-Natal/métodos , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Medicina Estatal , Trissomia/genética , Síndrome da Trissomia do Cromossomo 13/genética , Reino Unido
18.
Prenat Diagn ; 39(13): 1273-1282, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31671222

RESUMO

OBJECTIVE: To analyze the fetal fraction, fetal sex, and chromosomal aneuploidy in multiple pregnancies using noninvasive prenatal testing (NIPT). METHOD: A total of 362 pregnant women including 203 singleton pregnancies, 69 twins, and 90 higher-order multiple pregnancies were recruited. Fetal fractions estimated by size ratio-based and Y chromosome-based approaches in singleton pregnancies with male fetus were used as source data to establish the model. The model was then applied to multiple pregnancies for fetal fraction estimation. By comparing the fetal fractions estimated by size ratio to those estimated by Y chromosome or autosomal chromosomes, fetal sex and chromosomal aneuploidy can be analyzed. RESULTS: The size ratio-based approach has been well established in estimating fetal fractions for twin and higher-order multiple pregnancies. Fetal fraction had a positive correlation with gestational age in twin and triplet pregnancies. Fetal sex was determined with accuracies of 98.6% (95% CI, 92.19%-99.96%) in twins and 97.6% (95% CI, 91.76%-99.71%) in triplet pregnancies. Four trisomy 21, one trisomy 18, and one trisomy 13 cases were detected by NIPT. Two trisomy 21 singleton pregnancies and one trisomy 21 twin pregnancy were confirmed by karyotyping. CONCLUSION: Fetal sex and chromosomal aneuploidy in multiple pregnancies can be determined using NIPT.


Assuntos
Aneuploidia , Ácidos Nucleicos Livres/análise , Teste Pré-Natal não Invasivo , Gravidez Múltipla , Adolescente , Adulto , Feminino , Idade Gestacional , Humanos , Pessoa de Meia-Idade , Gravidez , Análise para Determinação do Sexo , Adulto Jovem
19.
Int J Mol Sci ; 20(22)2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31718077

RESUMO

The discovery of cell-free DNA (cfDNA) dates back to 1948, when Mandel and Metais found it in the sera of cancer patients [...].


Assuntos
Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/genética , DNA de Neoplasias/genética , Neoplasias/diagnóstico , Neoplasias/genética , Humanos
20.
Anal Bioanal Chem ; 411(29): 7725-7735, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760445

RESUMO

The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11-15 µm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization. Graphical abstract.


Assuntos
Doenças Cardiovasculares/metabolismo , Ácidos Nucleicos Livres/análise , Dispositivos Lab-On-A-Chip , Proteínas/análise , Autoanticorpos/análise , Biomarcadores/análise , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Microesferas , Proteínas/imunologia
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