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1.
Exp Suppl ; 111: 33-52, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588527

RESUMO

Molecular genetic methods have become an organic part of everyday clinical practice. In the past, molecular diagnostic tests were carried out for genetic diagnosis of a particular monogenic disease. In these situations the tests itself were used for identification of one particular genetic alteration (e.g., point mutation or deletion) of the gene of interest. Later, parallel with the development of the technology, the focus has shifted by allowing investigating at once targeted gene panels and even the whole exome/genome behind a suspected genetic disorder. Historically for these purposes, array-based methods (oligonucleotide arrays) and then next-generation sequencing-based methods have been used. High-throughput methods have been fundamentally transforming the everyday, routine genetic diagnostics, but older molecular techniques still have a role in clinical genetics. Here, we summarize the most important molecular genetic methods and shed light to the advantages and disadvantages of their application in routine diagnostics. We mainly focus on methods used for detection of germline alterations.


Assuntos
Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase em Tempo Real , Exoma , Humanos , Mutação
2.
J Med Microbiol ; 68(11): 1622-1628, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31596198

RESUMO

Introduction. Nosocomial transmission of Mycobacterium tuberculosis is an important health issue and the detection of tuberculosis (TB) cases is the main tool for controlling this disease.Aim. We aimed to assess the possible occurrence of nosocomial transmission of M. tuberculosis in a reference hospital for HIV/AIDS patients and evaluate both the performance of the Xpert MTB/RIF (Xpert) platform and drug resistance profiles.Methodology. We evaluated the performance of the Xpert platform. Samples that tested positive on the BACTEC MGIT 320 (MGIT320) platform were submitted for genotyping and drug susceptibility testing.Results. In this study, pulmonary and extrapulmonary samples from 407 patients were evaluated, and among these, 15.5 % were diagnosed with TB by the MGIT320 platform, with a TB/HIV coinfection rate of 52.4 %. The Xpert platform gave positive results for TB for 11 samples with negative results on the MGIT320 platform. In the genotyping results, 53.3 % of the strains clustered; of these strains, half were in two of the four clusters formed, and the patients had visited the hospital on the same day. Drug resistance was observed in 11.7 % of the strains.Conclusion. Putative nosocomial transmission of M. tuberculosis was detected, showing that genotyping is a powerful approach for understanding the dynamics of M. tuberculosis transmission, especially in a high-burden TB and HIV landscape.


Assuntos
Infecções por HIV/complicações , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/microbiologia , Síndrome de Imunodeficiência Adquirida/complicações , Antibióticos Antituberculose/farmacologia , Técnicas de Laboratório Clínico , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/microbiologia , Estudos Transversais , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Filogenia , Tuberculose , Tuberculose Pulmonar/diagnóstico
3.
Pan Afr Med J ; 33: 110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31489088

RESUMO

Introduction: The World Health Organization endorsed (2010) the use of Xpert MTB/RIF and countries are shifting from smear microscopy (smear)-based to Xpert MTB/RIF-based tuberculosis (TB) diagnostic algorithms. As with smear, sputum quality may predict the likelihood of obtaining a bacteriologically-confirmed TB when using Xpert MTB/RIF. Methods: From 08/12-11/2014, all people living with HIV were recruited at 22 clinics. For patients screened positive using the four TB symptoms their sputa were tested by Xpert MTB/RIF and smear. Laboratorians assessed and recorded sputum appearance and volume. The yield of bacteriologically-positive sputum evaluated using Xpert MTB/RIF and smear, likelihood-ratios were calculated. Results: Among 6,041 patients enrolled 2,296 were presumptive TB, 1,305 (56.8%) had > 1 sputa collected and 644/1,305 (49.3%) had both Xpert MTB/RIF and smear results. Since >1 sputa collected from 644 patients 954 sputa were tested by Xpert MTB/RIF and smear. Bacteriologically-positive sputum was two-fold higher with Xpert MTB/RIF 11.4% versus smear 5.3%, p < 0.001. Sputum appearance and quantity were not predictive of bacteriologically-positive results, except volume of 2ml to < 3ml, tested by Xpert MTB/RIF LR+= 1.26 (95% CI, 1.05-1.50). Conclusion: Xpert MTB/RIF test yield to bacteriologically-positive sputum was superior to smear. Sputum quality and quantity, however, were not consistently predictive of bacteriologically-positive results by Xpert MTB/RIF or smear.


Assuntos
Técnicas Bacteriológicas/métodos , Microscopia/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Adulto , Feminino , Seguimentos , Humanos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Tuberculose/epidemiologia
4.
Presse Med ; 48(7-8 Pt 1): 816-824, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31439443

RESUMO

Diagnosis of mature B cell malignancies is highly multidisciplinary. Biological tools provide diagnostic, prognostic and theranostic information. Biological hematology allows considering mature B cell diseases from two perspectives : cellular and molecular approaches. Cytomorphology and flow cytometry are tools from cell hematology. Conventional cytogenetics, FISH and molecular biology are tools from molecular hematology. NGS is a new technique that could dramatically change diagnostic and therapeutic management of B cell malignancies in the near future. Integration of clinical, pathological and biological data allows for personalized management of these diseases.


Assuntos
Técnicas de Laboratório Clínico/métodos , Leucemia de Células B/diagnóstico , Linfoma de Células B/diagnóstico , Integração de Sistemas , Hibridização Genômica Comparativa/métodos , Citodiagnóstico/métodos , Análise Citogenética/métodos , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunofenotipagem/métodos , Hibridização in Situ Fluorescente/métodos , Leucemia de Células B/genética , Leucemia de Células B/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Técnicas de Diagnóstico Molecular/métodos , Imagem Multimodal/métodos , Imagem Multimodal/tendências
5.
Presse Med ; 48(7-8 Pt 1): 832-841, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31444019

RESUMO

Lymphoplasmocytic lymphona with monoclonal lgM, rare. Median age at diagnosis 70 years old, frail population. Heterogenous clinic presentation. Molecular diagnosis with MYD88. Treatment required for symptomatic WM patients only. 1st line therapy: DRC. Input of targeted therapies (ibrutinib) for frail patients, maintenance effect.


Assuntos
Macroglobulinemia de Waldenstrom , Idade de Início , Idoso , Análise Mutacional de DNA/métodos , Idoso Fragilizado , Humanos , Técnicas de Diagnóstico Molecular/métodos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Fator 88 de Diferenciação Mieloide/genética , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/epidemiologia , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/terapia
6.
Presse Med ; 48(7-8 Pt 1): 850-858, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31447334

RESUMO

Follicular lymphoma, the second most common lymphoma, is characterized by its slow growth and is often considered incurable in advanced stages. Progresses in biology have contributed to better understand the complex and successive mechanisms of development of this pathology, whose diagnosis is based on a lymph node biopsy. However, the prognosis of the patients is heterogeneous and several indexes have been proposed to identify groups of patients with a similar life expectancy, in order to guide the therapeutic choices. The treatment has been modified in the last 20 years by the emergence of anti-CD20 monoclonal antibodies which constitute, alone or in combination, of the cornerstone of therapeutic management. After staging using, in particular, 18-fluorodeoxyglucose positron emission tomography, the therapeutic strategy will be adapted for each patient, ranging from simple watchful waiting to a combination of chemotherapy and anti-CD20 antibodies. Relapses (which often require a new lymph node biopsy to eliminate a possible histological transformation into an aggressive lymphoma with poorer prognosis) remain common but are still accessible to effective therapeutic interventions. Thanks to these advances, the median life expectancy of patients with follicular lymphoma now exceeds 15 years.


Assuntos
Linfoma Folicular , Biópsia , Diagnóstico Diferencial , Humanos , Imunofenotipagem/métodos , Imunoterapia/métodos , Imunoterapia/tendências , Expectativa de Vida , Linfoma Folicular/diagnóstico , Linfoma Folicular/epidemiologia , Linfoma Folicular/patologia , Linfoma Folicular/terapia , Técnicas de Diagnóstico Molecular , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/terapia , Medicina de Precisão/métodos , Medicina de Precisão/tendências , Prognóstico , Taxa de Sobrevida , Terapias em Estudo/métodos , Terapias em Estudo/tendências
8.
BMC Infect Dis ; 19(1): 591, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286879

RESUMO

BACKGROUND: We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland. CASE PRESENTATION: The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days. CONCLUSIONS: This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.


Assuntos
Infecções por Bunyaviridae , Epididimite , Meningite Viral , Metagenômica/métodos , Vírus da Febre do Flebótomo Napolitano , Adulto , Anticorpos Antivirais/sangue , Infecções por Bunyaviridae/diagnóstico , Infecções por Bunyaviridae/imunologia , Infecções por Bunyaviridae/virologia , Epididimite/diagnóstico , Epididimite/imunologia , Epididimite/virologia , Humanos , Itália , Masculino , Meningite Viral/diagnóstico , Meningite Viral/imunologia , Meningite Viral/virologia , Técnicas de Diagnóstico Molecular , RNA Viral/genética , Vírus da Febre do Flebótomo Napolitano/genética , Vírus da Febre do Flebótomo Napolitano/imunologia , Análise de Sequência de RNA , Suíça , Adulto Jovem
9.
J Med Microbiol ; 68(9): 1324-1329, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355739

RESUMO

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001).Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.


Assuntos
Córnea/virologia , Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Primers do DNA/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
Plant Dis ; 103(9): 2237-2245, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31306089

RESUMO

Phakopsora pachyrhizi, the causal agent of soybean rust (SBR), is a global threat to soybean production. Since the discovery of SBR in the continental United States, quantitative polymerase chain reaction assays based on the internal transcribed spacer (ITS) ribosomal DNA locus were established for its rapid detection. However, insufficient data were initially available to test assays against factors that could give rise to misidentification. This study aimed to reevaluate current assays for (i) the potential for false-positive detection caused by nontarget Phakopsora species and (ii) the potential for false-negative detection caused by intraspecific variation within the ITS locus of P. pachyrhizi. A large amount of intraspecific and intragenomic variation in ITS was detected, including the presence of polymorphic ITS copies within single leaf samples and within single rust sori. The diagnostic assays were not affected by polymorphisms in the ITS region; however, current assays are at risk of false positives when screened against other species of Phakopsora. This study raises caveats to the use of multicopy genes (e.g., ITS) in single-gene detection assays and discusses the pitfalls of inferences concerning the aerobiological pathways of disease spread made in the absence of an evaluation of intragenomic ITS heterogeneity.


Assuntos
Variação Genética , Phakopsora pachyrhizi , Doenças das Plantas , DNA Espaçador Ribossômico/genética , Técnicas de Diagnóstico Molecular/normas , Patologia Molecular , Phakopsora pachyrhizi/genética , Doenças das Plantas/microbiologia , Soja/microbiologia , Estados Unidos
11.
Arch Virol ; 164(10): 2581-2584, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359148

RESUMO

Maize chlorotic mottle virus (MCMV), an important quarantine virus, causes lethal necrosis in maize when coinfected with a potyvirid, which is seriously threatening the production of maize worldwide. In this study, recombinase polymerase amplification (RPA), a novel isothermal DNA amplification and detection technique, was developed to detect MCMV in maize crops. A pair of specific primers was designed based on the conserved sequences of the MCMV coat protein region. The RT-RPA assay was carried out as an isothermal reaction at 38 °C that was complete within 30 min, and no cross-reactivity was detected with other viruses infecting maize in China. The limit of detection of the RT-RPA assay was tenfold lower than that of ordinary RT-PCR. Moreover, this method was successfully applied to test field-collected samples. The newly developed RT-RPA assay offers a reliable, sensitive and efficient method for rapid detection of MCMV in maize in equipment-limited diagnostic laboratories and on-site facilities.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , Tombusviridae/isolamento & purificação , Proteínas do Capsídeo/genética , China , Primers do DNA/genética , Sensibilidade e Especificidade , Temperatura Ambiente , Fatores de Tempo , Tombusviridae/classificação , Tombusviridae/genética
12.
J Cancer Res Clin Oncol ; 145(8): 1977-1986, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31309300

RESUMO

CONTEXT: Parathyroid carcinoma (PC) is a rare endocrine malignancy with no approved systemic therapies for unresectable locally invasive or distant metastatic disease. Understanding the molecular changes in advanced PC can provide better understanding of this disease and potentially help directing targeted therapy. OBJECTIVE: To evaluate tumor-specific genetic changes using next-generation sequencing (NGS) panels. DESIGN: All patients with advanced PC were tested for hot-spot panels using NGS panels including a 50-gene panel, a 409-gene panel if the standard 50-gene panel (Ion Torrent, Life Technology) was negative or a FoundationOne panel. SETTING: The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. PATIENTS OR OTHER PARTICIPANTS: 11 patients with advanced PC were selected to undergo molecular testing. MAIN OUTCOME MEASURE(S): Genetic profiles of advanced PC. RESULTS: Among the 11 patients, 4 patients had the 50-gene panel only, 6 had 409-gene panel after a negative 50-gene panel and 1 had FoundationOne. One patient who had 50-gene panel only also had his metastatic site (esophagus) of his tumor tested with FoundationOne. The most common mutations identified were in the PI3 K (PIK3CA, TSC1 and ATM) (4/11 patients) and TP53 (3/11) pathways. Genes not previously reported to be mutated in PC included: SDHA, TERT promoter and DICER1. Actionable mutations were found in 54% (6/11) of the patients. CONCLUSIONS: Mutational profiling using NGS panels in advanced PC has yielded important potentially targetable genetic alterations. Larger studies are needed to identify commonly mutated genes in advanced PC patients. Development of novel therapies targeting these cellular pathways should be considered.


Assuntos
Carcinoma/genética , Perfilação da Expressão Gênica , Técnicas de Diagnóstico Molecular/métodos , Monitorização Fisiológica/métodos , Neoplasias das Paratireoides/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/diagnóstico , Carcinoma/patologia , Carcinoma/terapia , Análise Mutacional de DNA/métodos , Progressão da Doença , Feminino , Seguimentos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/tendências , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Neoplasias das Paratireoides/diagnóstico , Neoplasias das Paratireoides/patologia , Neoplasias das Paratireoides/terapia
13.
Dis Colon Rectum ; 62(7): 859-866, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31188187

RESUMO

BACKGROUND: Vascular Ehlers-Danlos syndrome is a rare and severe genetic condition leading to spontaneous, potentially life-threatening arterial and digestive complications. Colonic ruptures are a common feature of the disease, but clear recommendations on their management are lacking. OBJECTIVE: This study aimed to identify surgery-related morbidity and 30-day postoperative mortality after colonic perforation. DESIGN: This was a retrospective review. SETTING: A large cohort of patients with vascular Ehlers-Danlos syndrome was followed in a tertiary referral center. PATIENTS: Between 2000 and 2016, the French National Reference Centre for Rare Vascular Diseases (HEGP, AP-HP, Paris, France) followed 148 patients with molecularly proven vascular Ehlers-Danlos syndrome. MAIN OUTCOME MEASURES: The primary outcomes measured were surgery-related morbidity and 30-day postoperative mortality. RESULTS: Of 133 patients with molecularly proven vascular Ehlers-Danlos syndrome, 30 (22%) had a history of colonic perforation and 15 (50%) were males. These subjects were diagnosed with vascular Ehlers-Danlos syndrome at a younger age than patients with a history of GI events without colonic perforation (p = 0.0007). There were 46 colonic perforations, median 1.0 event per patient (interquartile range, 1.0-2.0). Reperforations occurred in 14 (47%) patients, mostly males. Surgical management consisted of Hartmann procedures or subtotal abdominal colectomies, with a nonnegligible rate of reperforation following partial colonic resection (11 patients, 41%). LIMITATIONS: The main limitations of this work are its retrospective design and that the diagnosis of vascular Ehlers-Danlos syndrome was made after colonic perforations in a majority of patients. CONCLUSION: Colonic perforations seem more severe in males, with a high rate of reperforation after Hartmann procedure. Subtotal colectomy may reduce digestive morbidity, particularly in male patients. Additional studies are required to identify other predictors of reperforation. See Video Abstract at http://links.lww.com/DCR/A937.


Assuntos
Doenças do Colo/etiologia , Doenças do Colo/cirurgia , Síndrome de Ehlers-Danlos/complicações , Perfuração Intestinal/etiologia , Perfuração Intestinal/cirurgia , Complicações Pós-Operatórias/etiologia , Adolescente , Adulto , Criança , Colectomia/efeitos adversos , Colectomia/mortalidade , Síndrome de Ehlers-Danlos/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Recidiva , Estudos Retrospectivos , Adulto Jovem
14.
APMIS ; 127(9): 627-634, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31225920

RESUMO

Sexually transmitted infections (STIs) remain major public health problems globally. Appropriate laboratory diagnosis of STIs is rare in Ukraine. We investigated the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) using the US FDA-approved Aptima Combo 2 and Aptima TV assays and compared the results with the conventional routine diagnostic tests (CDTs) in Ukraine. Urogenital swabs from consecutive mostly symptomatic females (n = 296) and males (n = 159) were examined. The prevalences were as follows: 10% (n = 47) of TV, 5.3% (n = 24) of CT and 1.5% (n = 7) of NG. The specificity of some CDTs was high, for example, 100% for NG culture, TV IgG ELISA, CT IgM ELISA and CT microscopy, but lower for other CDTs, that is, from 44% to 99.8%. The sensitivity of all CDTs was suboptimal, that is, 71% (n = 5) for NG microscopy, 57% (n = 4) for NG culture, 53% (n = 8) for CT IgG ELISA, 33% (n = 1) for TV IgG ELISA, 28% (n = 13) for TV microscopy, 25% (n = 1) for CT IgA ELISA, 20% (n = 3) for CT IgM ELISA and 0% (n = 0) for CT microscopy. The prevalences of particularly TV and CT were high, but substantial also for NG, in Ternopil, Ukraine. The sensitivities of all CDTs were low, and widespread implementation of validated, quality-assured and cost-effective molecular diagnostic STI tests in Ukraine is imperative.


Assuntos
Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/epidemiologia , Gonorreia/diagnóstico , Gonorreia/epidemiologia , Vaginite por Trichomonas/diagnóstico , Vaginite por Trichomonas/epidemiologia , Adolescente , Adulto , Chlamydia trachomatis/isolamento & purificação , Estudos Transversais , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Neisseria gonorrhoeae/isolamento & purificação , Prevalência , Sensibilidade e Especificidade , Doenças Sexualmente Transmissíveis/diagnóstico , Doenças Sexualmente Transmissíveis/epidemiologia , Trichomonas vaginalis/isolamento & purificação , Ucrânia/epidemiologia , Estados Unidos , United States Food and Drug Administration , Adulto Jovem
15.
J Med Microbiol ; 68(8): 1204-1210, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31184572

RESUMO

INTRODUCTION: In recent years, metagenomic next-generation sequencing (mNGS) has become widely used in medical microbiology to detect pathogen infection. AIM: We aimed to assess the diagnostic performance of mNGS of cerebrospinal fluid (CSF) for prediction of cryptococcal meningitis (CM). METHODOLOGY: A comparative evaluation of mNGS (performed on CSF samples) and conventional methods, including India ink staining, culture for fungi and cryptococcal-antigen (CrAg) detection by enzyme immunoassay, was performed on 12 consecutive non-HIV-infected patients with chronic or subacute CM. RESULTS: India ink staining and culture of the CSF were positive for Cryptococcus in 83.33 % (10/12) of the samples; 100 % (11/11) were positive via CrAg EIA. The mNGS results of the CSF identified DNA sequences corresponding to Cryptococcus in 75 % of samples (9/12). However, the DNA of both C. neoformans s.l. and C. gattii s.l. was detected concurrently in 33.33 % (4/12). CONCLUSION: mNGS is helpful for identifying Cryptococcus species. The application of mNGS, together with India ink staining, culture methods, and CrAg, may significantly improve the diagnostic precision in CM, thereby informing choice of appropriate antifungal treatment courses.


Assuntos
Cryptococcus gattii/genética , Cryptococcus neoformans/genética , Meningite Criptocócica/diagnóstico , Meningite Criptocócica/microbiologia , Metagenômica , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Idoso , Coinfecção/diagnóstico , Cryptococcus gattii/isolamento & purificação , Cryptococcus neoformans/isolamento & purificação , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Masculino , Meningite Criptocócica/líquido cefalorraquidiano , Técnicas Microbiológicas , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Análise de Sequência de DNA
16.
J Med Microbiol ; 68(8): 1219-1226, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237534

RESUMO

PURPOSE: The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles. METHODOLOGY: A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell. RESULTS: All GN bacteria identifications made by MinION sequencing for 30 min using the What's In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum ß-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes. CONCLUSION: We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.


Assuntos
Bacteriemia/microbiologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Genoma Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Nanoporos , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/normas , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Fatores de Tempo
17.
Clin Cancer Res ; 25(15): 4586-4588, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31160470

RESUMO

Multitarget assays have become common in clinical molecular diagnostic laboratories. However, all assays, no matter how well designed, have inherent gaps due to technical and biological limitations. In some clinical cases, testing by multiple methodologies is needed to address these gaps and ensure the most accurate molecular diagnoses.See related article by Benayed et al., p. 4712.


Assuntos
Técnicas de Diagnóstico Molecular
19.
J Clin Pathol ; 72(8): 513-519, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154423

RESUMO

The importance of circular RNAs (circRNAs) in pathological processes like cancer is evident. Among the circRNAs, recent studies have brought circPVT1 under focus as the most potent oncogenic non-coding RNA. Recent studies on various aspects of circPVT1, including its biogenesis, molecular alteration and its probable role in oncogenesis, have been conducted for research and clinical interest. In this review, a first attempt has been made to summarise the available data on circPVT1 from PubMed and other relevant databases with special emphasis on its role in development, progression and prognosis of various malignant conditions. CircPVT1 is derived from the same genetic locus encoding for long non-coding RNA lncPVT1; however, existing literature suggested circPVT1 and lncPVT1 are transcripted independently by different promoters. The interaction between circRNA and microRNA has been highlighted in majority of the few malignancies in which circPVT1 was studied. Besides its importance in diagnostic and prognostic procedures, circPVT1 seemed to have huge therapeutic potential as evident from differential drug response of cancer cell line as well as primary tumors depending on expression level of the candidate. circPVT1 in cancer therapeutics might be promising as a biomarker to make the existing treatment protocol more effective and also as potential target for designing novel therapeutic intervention.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , RNA/genética , Animais , Biomarcadores Tumorais/biossíntese , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas de Diagnóstico Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Valor Preditivo dos Testes , Prognóstico , RNA/biossíntese , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/metabolismo
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