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1.
Nat Commun ; 11(1): 4774, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963224

RESUMO

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.


Assuntos
Líquidos Corporais/química , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Sepse/diagnóstico , Candida/genética , Candida/isolamento & purificação , Candidíase/diagnóstico , Contagem de Colônia Microbiana , Doenças Transmissíveis/diagnóstico , DNA/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Reação em Cadeia da Polimerase/métodos , RNA/isolamento & purificação , Sensibilidade e Especificidade , Sepse/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Urina
2.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
3.
Zhonghua Liu Xing Bing Xue Za Zhi ; 41(6): 913-918, 2020 Jun 10.
Artigo em Chinês | MEDLINE | ID: mdl-32564559

RESUMO

Objectives: To understand the prevalence of HIV nucleic acid using internet-based dry blood spots HIV testing strategy in men who had sex with men (MSM) and to probe the factors associated with HIV infection. Methods: Using convenient sampling method, 1 375 MSM were recruited and their dry blood spots samples were collected before being mailed to the laboratories for HIV nucleic acid testing. Results were showed to these MSM on a specific website by inputting their codes to it. Non-conditional binary logistic regression method was used to identify the associated factors on HIV infection. Results: The overall proportions of HIV nucleic acid positives appeared as 9.7% (131/1 349) and HIV antibody positives as 8.3% (112/1 349). Fresh infections accounted for 14.5% (19/131) among the newly-identified HIV nucleic acid positives, and the interval was ranging from 6 to 120 days, between the laboratory testings and the closest date that experiencing high risk behavior. Risk factors that related to HIV infection would include: 30 to 39 years of age (comparing to those under the age of 30, OR=1.88, 95%CI: 1.07-3.29), ≥8 000 Yuan of monthly income (comparing to those without income, OR=0.42, 95%CI: 0.19-0.96), inconsistent condom use during anal sexual contacts in the last six months (compared with those who had not anal sex or used condoms consistently in anal sex in the past six months, OR=2.22, 95%CI: 1.45-3.40), ever use of Rush Poppers (compared with those who never used Rush Poppers, OR=2.33, 95%CI: 1.49-3.64), addictive drug abuse (compared with those who never abused addictive drugs, OR=5.43, 95%CI: 2.32-12.69), and not having regular sexual partners (compared with having regular sexual partners, OR=1.74, 95%CI: 1.13-2.68) etc.. Conclusions: Dry blood spots HIV nucleic acid testing could help to identify the fresh HIV infections at an early stage, so as to prevent further transmission in the MSM population, among which fresh HIV infections accounted for a fairly large proportion. It is necessary to set up programs in reducing the abuse of drugs or Rush Poppers, and to promote condom use and advocate on stable sexual partnership etc., among the MSM population.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Infecções por HIV/epidemiologia , Homossexualidade Masculina/estatística & dados numéricos , Internet , Ácidos Nucleicos/sangue , Adulto , Pequim/epidemiologia , Estudos de Viabilidade , Humanos , Masculino , Fatores de Risco
4.
Biosens Bioelectron ; 151: 111998, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999593

RESUMO

The recent outbreaks of mosquito-borne diseases (e.g., zika, dengue, and chikungunya) increased public health burden in developing countries. To control the spread of these infectious diseases, a simple, economic, reliable, sensitive, and selective diagnostic platform is required. Considering demand for affordable and accessible methods, we have demonstrated a two-step strategy for extraction and detection of viral RNAs of infectious diseases within 1 h. Ready-to-use devices for viral RNA extraction and detection were successfully fabricated using paper as a substrate. Viral RNA (e.g., zika, dengue, and chikungunya) was captured and eluted using a handheld RNA extraction paper-strip device, and another paper-chip device was used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a detection limit of a single copy and 10 copies of viral RNA in phosphate buffer solution (PBS) and serum, respectively. With these proposed devices, we have detected viral RNAs of zika and dengue in clinical human serum samples. The proposed paper-based extraction and detection platforms could be employed for detection of infectious viral diseases from complex clinical samples in resource-limited settings.


Assuntos
Técnicas Biossensoriais , Culicidae/virologia , Ácidos Nucleicos/sangue , Vírus de RNA/isolamento & purificação , Animais , Diagnóstico Precoce , Humanos , Ácidos Nucleicos/química , Papel , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Zika virus/genética , Zika virus/isolamento & purificação , Zika virus/patogenicidade , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/genética , Infecção por Zika virus/virologia
5.
Talanta ; 209: 120581, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892020

RESUMO

Conjugated polyelectrolytes (CPEs) have been widely used as reporters in colorimetric assays targeting nucleic acids. CPEs provide naked eye detection possibility by their superior optical properties however, as concentration of target analytes decrease, trace amounts of nucleic acid typically yield colorimetric responses that are not readily perceivable by naked eye. Herein, we report a pixelated analysis approach for correlating colorimetric responses of CPE with nucleic acid concentrations down to 1 nM, in plasma samples, utilizing a smart phone with an algorithm that can perform analytical testing and data processing. The detection strategy employed relies on conformational transitions between single stranded nucleic acid-cationic CPE duplexes and double stranded nucleic acid-CPE triplexes that yield distinct colorimetric responses for enabling naked eye detection of nucleic acids. Cationic poly[N,N,N-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide] is utilized as the CPE reporter deposited on a polyvinylidene fluoride (PVDF) membrane for nucleic acid assay. A smart phone application is developed to capture and digitize the colorimetric response of the individual pixels of the digital images of CPE on the PVDF membrane, followed by an analysis using the algorithm. The proposed pixelated approach enables precise quantification of nucleic acid assay concentrations, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses, for instance, RGB analysis. The obtained results illustrate that a ubiquitous smart phone could be utilized for point of care colorimetric nucleic acids assays in complex matrices without requiring sophisticated software or instrumentation.


Assuntos
Colorimetria/métodos , Ácidos Nucleicos/sangue , Polieletrólitos/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Cátions/química , Colorimetria/instrumentação , Desenho de Equipamento , Humanos , Ácidos Nucleicos/análise , Papel , Sistemas Automatizados de Assistência Junto ao Leito , Polímeros/química , Polivinil/química , Smartphone , Tiofenos/química
6.
ACS Appl Mater Interfaces ; 12(2): 2095-2106, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31845577

RESUMO

Spinal cord injury (SCI), began with a primary injury including contusion and compression, is a common disease caused by various pathogenesis. Characterized disruption of axons and irreversible loss of neurons in SCI, and further damage in spinal cord tissue caused by following secondary injuries, such as the formation of glial scar and inflammation, makes it even harder to recover for affected patients. Tetrahedral framework nucleic acid (tFNA), which possesses the capability of promoting neuroprotection and neuroregeneration in vitro, might alleviate the injuries, and facilitate the neural tissue regeneration in experimental animal models of SCI. Here, we developed a concomitant treatment of tFNA and neural stem cells (NSCs) for the synergistic therapy in treating the injury of the spinal cord. We first observed that tFNA could promote cell proliferation of NSCs then verified that the concomitant treatment of tFNA and NSCs showed the neuroprotective actions by increasing the survival of transplanted NSCs. Furthermore, the recovery of motor function and the tissue regeneration in the lesion site of the spinal cord achieved the best performance in the SCI rats treated with the combination of tFNA and NSCs than others, and the formation of glial scar was the least. Our findings provide novel evidence of a promising strategy for synergistic treatment of SCI in the future.


Assuntos
Regeneração Nervosa , Células-Tronco Neurais/transplante , Ácidos Nucleicos/uso terapêutico , Traumatismos da Medula Espinal/terapia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Atividade Motora , Proteínas do Tecido Nervoso/metabolismo , Ácidos Nucleicos/sangue , Ratos , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/fisiopatologia
7.
Sensors (Basel) ; 19(23)2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31795306

RESUMO

Information about the characteristics of measuring chips according to their storage conditions is of great importance for clinical diagnosis. In our present work, we have studied the capability of chips to detect nanowire biosensors when they are either freshly prepared or have been stored for either one or two years in a clean room. Potential to detect DNA oligonucleotides (oDNAs)-synthetic analogues of microRNAs (miRNAs) 198 and 429 that are associated with the development of prostate cancer (PCa)-in buffer solution was demonstrated using a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). To provide biospecific detection, nanowire surfaces were sensitized with oligonucleotide probes (oDNA probes) complimentary to the known sequences of miRNA 183 and 484. In this study it is demonstrated that freshly prepared SOI-NW biosensor chips with n-type conductance and immobilized oDNA probes exhibit responses to the addition of complimentary oDNAs in buffer, leading to decreases in chips' conductance at a concentration of 3.3 × 10-16 M. The influence of storage time on the characteristics of SOI-NW biosensor chips is also studied herein. It is shown that a two-year storage of the chips leads to significant changes in their characteristics, resulting in "inverse" sensitivity toward negatively charged oDNA probes (i.e., through an increase in chips' conductance). It is concluded that the surface layer makes the main contribution to conductance of the biosensor chip. Our results indicate that the detection of target nucleic acid molecules can be carried out with high sensitivity using sensor chips after long-term storage, but that changes in their surface properties, which lead to inversed detection signals, must be taken into account. Examples of the applications of such chips for the detection of cancer-associated microRNAs in plasma samples of patients with diagnosed prostate cancer are given. The results obtained herein are useful for the development of highly sensitive nanowire-based diagnostic systems for the revelation of (prostate) cancer-associated microRNAs in human plasma.


Assuntos
Biomarcadores Tumorais/sangue , Técnicas Biossensoriais/métodos , MicroRNAs/sangue , Neoplasias da Próstata/sangue , Humanos , Masculino , Nanofios/química , Ácidos Nucleicos/sangue , Ácidos Nucleicos/isolamento & purificação , Silício/química
8.
Lab Chip ; 19(22): 3853-3861, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31621762

RESUMO

We report a bifurcated continuous field-flow fractionation (BCFFF) chip for high-yield and high-throughput (20 min) extraction of nucleic acids from physiological samples. The design uses a membrane ionic transistor to sustain low-ionic strength in a localized region at a junction, such that the resulting high field can selectively isolate high-charge density nucleic acids from the main flow channel and insert them into a standardized buffer in a side channel that bifurcates from the junction. The high local electric field and the bifurcated field-flow design facilitate concentration reduction of both divalent cation (Ca2+) and molecular PCR inhibitors by more than two orders of magnitude, even with high-throughput continuous loading. The unique design with a large (>20 mM mm-1) on-chip ionic-strength gradient allows miniaturization into a high-throughput field-flow fractionation chip that can be integrated with upstream lysing and downstream PCR/sensor modules for various nucleic acid detection/quantification applications. A concentration-independent 85% yield for extraction and an overall post-PCR yield exceeding 60% are demonstrated for a 111 bp dsDNA in 10 µL of human plasma, compared to no amplification with the raw sample. A net yield four times larger than a commercial extraction kit is demonstrated for miR-39 in human plasma.


Assuntos
Fracionamento por Campo e Fluxo , Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas , Ácidos Nucleicos/isolamento & purificação , Cálcio/sangue , Fracionamento por Campo e Fluxo/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Ácidos Nucleicos/sangue , Ácidos Nucleicos/genética , Reação em Cadeia da Polimerase
9.
Free Radic Biol Med ; 145: 336-341, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31586654

RESUMO

BACKGROUND: The oxidized guanine nucleosides, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), derived from DNA and RNA, respectively, were used to investigate the importance of oxidative stress to nucleic acids in vivo. High urinary excretion of 8-oxodG is associated with cancer development, whereas high urinary excretion of 8-oxoGuo is associated with mortality in type 2 diabetes. Like creatinine, these small water-soluble molecules are not reabsorbed in the kidney. Therefore, 8-oxo nucleoside/creatinine reciprocal concentration ratios are identical in plasma and urine. The total amount of 8-oxo guanine nucleosides excreted by the kidneys is the product of plasma concentration and glomerular filtration rate. METHODS: With relevant equations and an estimated glomerular filtration rate, the 24-h urinary excretion of 8-oxodG and 8-oxoGuo was calculated in 2679 subjects with type 2 diabetes, displaying good correlation with the measured urinary 8-oxo nucleoside/creatinine ratio: DNA oxidation r = 0.86 and RNA oxidation r = 0.84 (p < 0.05 for both). RESULTS: Survival analyses based on the quartiles of the 8-oxodG/creatinine ratio and the quartiles of calculated 24-h urinary excretion rate of the 2679 subjects gave similar hazard ratio estimates for death due to all causes. This finding was similar for the 8-oxoGuo hazard ratio estimates. CONCLUSIONS: This study shows that oxidatively generated modifications to DNA and RNA in vivo can be measured using 1) a spot urine sample, normalized to urinary creatinine, 2) 24-h urine, or 3) a single plasma sample based on concentrations of 8-oxo nucleoside and creatinine and glomerular filtration rate.


Assuntos
Biomarcadores , Neoplasias , 8-Hidroxi-2'-Desoxiguanosina/sangue , 8-Hidroxi-2'-Desoxiguanosina/urina , Biomarcadores/sangue , Biomarcadores/urina , Dano ao DNA , Humanos , Neoplasias/sangue , Neoplasias/urina , Ácidos Nucleicos/sangue , Ácidos Nucleicos/química , Ácidos Nucleicos/urina , Estresse Oxidativo/genética , Modelos de Riscos Proporcionais
10.
Free Radic Biol Med ; 145: 256-283, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31563634

RESUMO

Oxidative stress is associated with the development and progression of numerous diseases. However, targeting oxidative stress has not been established in the clinical management of any disease. Several methods and markers are available to measure oxidative stress, including direct measurement of free radicals, antioxidants, redox balance, and oxidative modifications of cellular macromolecules. Oxidatively generated nucleic acid modifications have attracted much interest due to the pre-mutagenic oxidative modification of DNA into 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), associated with cancer development. During the last decade, the perception of RNA has changed from that of a 'silent messenger' to an 'active contributor', and, parallelly oxidatively generated RNA modifications measured as 8-oxo-7,8-dihydro-guanosine (8-oxoGuo), has been demonstrated as a prognostic factor for all-caused and cardiovascular related mortality in patients with type 2 diabetes. Several attempts have been made to modify the amount of oxidative nucleic acid modifications. Thus, this review aims to introduce researchers to the measurement of oxidatively generated nucleic acid modifications as well as critically review previous attempts and provide future directions for targeting oxidatively generated nucleic acid modifications.


Assuntos
Biomarcadores , Neoplasias , Ácidos Nucleicos/genética , Estresse Oxidativo/genética , 8-Hidroxi-2'-Desoxiguanosina/sangue , 8-Hidroxi-2'-Desoxiguanosina/urina , Biomarcadores/sangue , Biomarcadores/urina , Dano ao DNA/genética , Humanos , Neoplasias/sangue , Neoplasias/urina , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina
11.
Acta Trop ; 199: 105120, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31376368

RESUMO

Chagas disease has become a global health problem due to migration of infected people out of Latin America to non-endemic countries. For more than 40 years, only the nitroimidazole compounds Benznidazole and Nifurtimox, have been used for specific treatment of Trypanosoma cruzi infection with disappointing results, specially due to the long duration of treatment and adverse events in the chronic phase. In the last years, ergosterol inhibitors have been also proposed for specific treatment. Different randomized clinical trials were performed for evaluating their treatment efficacy and safety. One of the greatest concerns in clinical trials is to provide an early surrogate biomarker of response to trypanocidal chemotherapy. Serological response is slow and the classical parasitological tests have poor sensitivity and are time-consuming. Nowadays, PCR is the most helpful tool for assessing treatment response in a short period of time. Different protocols of PCR have been developed, being quantitative real time PCR based on amplification of repetitive satellite or minicircle DNA sequences plus an internal amplification standard, the mostly employed strategies in clinical trials. Standardized protocols and the use of an external quality assessment ensure adequate technical procedures and reliable data. Clinical trials have shown a significant reduction in parasite loads, reaching undetectable DNA levels in bloodstream after specific treatment, however events of treatment failure have also been reported. Treatment failure could be due to inadequate penetrance of the drugs into the affected tissues, to the presence of primary or secondary drug resistance of the infecting strains as well as to the existence of dormant parasite variants reluctant to drug action. The early diagnosis of drug resistance would improve clinical management of Chagas disease patients, allowing dictating alternative therapies with a combination of existing drugs or new anti-T. cruzi agents. The aim of this review was to describe the usefulness of detecting T.cruzi DNA by means of real time PCR assays, as surrogate biomarker in clinical trials for evaluating new drugs for CD or new regimens of available drugs and the possibility to detect treatment failure.


Assuntos
Doença de Chagas/terapia , Ácidos Nucleicos/análise , Reação em Cadeia da Polimerase em Tempo Real , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Biomarcadores , Doença de Chagas/parasitologia , Doença Crônica , Resistência a Medicamentos/genética , Humanos , Nifurtimox/farmacologia , Nifurtimox/uso terapêutico , Nitroimidazóis/farmacologia , Nitroimidazóis/uso terapêutico , Ácidos Nucleicos/sangue , Carga Parasitária , Falha de Tratamento , Resultado do Tratamento , Tripanossomicidas/farmacologia , Trypanosoma cruzi/genética
12.
J Virol Methods ; 273: 113715, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31419455

RESUMO

Detection and quantification of viral nucleic acids are important for diagnosing current viral infections and monitoring response to antiviral therapy. Automated nucleic acid extraction and purification platforms are routinely used during the first step in these processes in clinical and research laboratories. Here, we compare the extraction efficiencies of four MagNA Pure magnetic bead-based nucleic acid extraction platforms and associated kits using samples positive for nucleic acids from HAV, HBV, HCV, HDV, and HEV. These five hepatitis viruses are diverse in their virion structures and type of nucleic acid that compose their genomes. We found that the most efficient nucleic acid extraction platform and corresponding kit, when averaged across all tested viruses, was the MagNA Pure 96, which yielded twice as much detectable nucleic acid as the other platforms. However, the relative efficiencies of the different platforms varied by virus type, suggesting that an extraction platform that is more efficient for one virus type will not necessarily function better with a different virus type. Our results show that the choice of a nucleic acid extraction platform influences the sensitivity of the methodology and has the potential to generate false-negative results especially in samples with low levels of viral nucleic acids.


Assuntos
Genoma Viral , Vírus de Hepatite/isolamento & purificação , Ácidos Nucleicos/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , DNA Viral/isolamento & purificação , Hepatite Viral Humana , Humanos , Ácidos Nucleicos/sangue , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade
13.
Cochrane Database Syst Rev ; 8: CD011871, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31425612

RESUMO

BACKGROUND: Early diagnosis of leptospirosis may contribute to the effectiveness of antimicrobial therapy and early outbreak recognition. Nucleic acid and antigen detection tests have the potential for early diagnosis of leptospirosis. With this systematic review, we assessed the sensitivity and specificity of nucleic acid and antigen detection tests. OBJECTIVES: To determine the diagnostic test accuracy of nucleic acid and antigen detection tests for the diagnosis of human symptomatic leptospirosis. SEARCH METHODS: We searched electronic databases including MEDLINE, Embase, the Cochrane Library, and regional databases from inception to 6 July 2018. We did not apply restrictions to language or time of publication. SELECTION CRITERIA: We included diagnostic cross-sectional studies and case-control studies of tests that made use of nucleic acid and antigen detection methods in people suspected of systemic leptospirosis. As reference standards, we considered the microscopic agglutination test alone (which detects antibodies against leptospirosis) or in a composite reference standard with culturing or other serological tests. Studies were excluded when the controls were healthy individuals or when there were insufficient data to calculate sensitivity and specificity. DATA COLLECTION AND ANALYSIS: At least two review authors independently extracted data from each study. We used the revised Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2) to assess risk of bias. We calculated study-specific values for sensitivity and specificity with 95% confidence intervals (CI) and pooled the results in a meta-analysis when appropriate. We used the bivariate model for index tests with one positivity threshold, and we used the hierarchical summary receiver operating characteristic model for index tests with multiple positivity thresholds. As possible sources of heterogeneity, we explored: timing of index test, disease prevalence, blood sample type, primers or target genes, and the real-time polymerase chain reaction (PCR) visualisation method. These were added as covariates to the meta-regression models. MAIN RESULTS: We included 41 studies evaluating nine index tests (conventional PCR (in short: PCR), real-time PCR, nested PCR, PCR performed twice, loop-mediated isothermal amplification, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, immunochromatography-based lateral flow assay, and dipstick assay) with 5981 participants (1834 with and 4147 without leptospirosis). Methodological quality criteria were often not reported, and the risk of bias of the reference standard was generally considered high. The applicability of findings was limited by the frequent use of frozen samples. We conducted meta-analyses for the PCR and the real-time PCR on blood products.The pooled sensitivity of the PCR was 70% (95% CI 37% to 90%) and the pooled specificity was 95% (95% CI 75% to 99%). When studies with a high risk of bias in the reference standard domain were excluded, the pooled sensitivity was 87% (95% CI 44% to 98%) and the pooled specificity was 97% (95% CI 60% to 100%). For the real-time PCR, we estimated a summary receiver operating characteristic curve. To illustrate, a point on the curve with 85% specificity had a sensitivity of 49% (95% CI 30% to 68%). Likewise, at 90% specificity, sensitivity was 40% (95% CI 24% to 59%) and at 95% specificity, sensitivity was 29% (95% CI 15% to 49%). The median specificity of real-time PCR on blood products was 92%. We did not formally compare the diagnostic test accuracy of PCR and real-time PCR, as direct comparison studies were lacking. Three of 15 studies analysing PCR on blood products reported the timing of sample collection in the studies included in the meta-analyses (range 1 to 7 days postonset of symptoms), and nine out of 16 studies analysing real-time PCR on blood products (range 1 to 19 days postonset of symptoms). In PCR studies, specificity was lower in settings with high leptospirosis prevalence. Other investigations of heterogeneity did not identify statistically significant associations. Two studies suggested that PCR and real-time PCR may be more sensitive on blood samples collected early in the disease stage. Results of other index tests were described narratively. AUTHORS' CONCLUSIONS: The validity of review findings are limited and should be interpreted with caution. There is a substantial between-study variability in the accuracy of PCR and real-time PCR, as well as a substantial variability in the prevalence of leptospirosis. Consequently, the position of PCR and real-time PCR in the clinical pathway depends on regional considerations such as disease prevalence, factors that are likely to influence accuracy, and downstream consequences of test results. There is insufficient evidence to conclude which of the nucleic acid and antigen detection tests is the most accurate. There is preliminary evidence that PCR and real-time PCR are more sensitive on blood samples collected early in the disease stage, but this needs to be confirmed in future studies.


Assuntos
Anticorpos Antibacterianos/imunologia , Leptospira/imunologia , Leptospirose/diagnóstico , Ácidos Nucleicos/sangue , Reação em Cadeia da Polimerase/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Leptospirose/sangue , Curva ROC , Sensibilidade e Especificidade
14.
Proc Natl Acad Sci U S A ; 116(33): 16240-16249, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31358642

RESUMO

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Nanotecnologia/tendências , Ácidos Nucleicos/isolamento & purificação , Patologia Molecular/métodos , Proteínas Sanguíneas/química , Ensaio de Imunoadsorção Enzimática , Proteína do Núcleo p24 do HIV/sangue , Proteína do Núcleo p24 do HIV/isolamento & purificação , Proteína do Núcleo p24 do HIV/urina , Humanos , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Troponina I/sangue , Troponina I/isolamento & purificação
16.
Forensic Sci Med Pathol ; 15(3): 332-341, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31054024

RESUMO

Postmortem diagnosis of extreme-weather-related deaths is a challenging forensic task. Here, we present a state-of-the-art study that employed attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy in combination with Chemometrics for postmortem diagnosis of fatal hypothermia/hyperthermia by biochemical investigation of plasma in rats. The results of principal component analysis (PCA) and spectral analysis revealed that plasma samples from the fatal hypothermia, fatal hyperthermia, and control groups, are substantially different from each other based on the spectral variations associated with the lipid, carbohydrate and nucleic acid components. Two partial least squares-discriminant analysis (PLS-DA) classification models (hypothermia-nonhypothermia and hyperthermia-nonhyperthermia binary models) with a 100% accuracy rate were constructed. Subsequently, internal cross-validation was performed to assess the robustness of these two models, which resulted in 98.1 and 100% accuracy. Ultimately, classification predictions of 42 unknown plasma samples were performed by these two models, and both models achieved 100% accuracy. Additionally, our results demonstrated that hemolysis and postmortem hypothermic/hyperthermic effects did not weaken the prediction ability of these two classification models. In summary, this work demonstrates ATR-FTIR spectroscopy's great potential for postmortem diagnosis of fatal hypothermia/hyperthermia.


Assuntos
Febre/diagnóstico , Hipotermia/diagnóstico , Plasma/química , Espectroscopia de Infravermelho com Transformada de Fourier , Animais , Biomarcadores/sangue , Glicemia/análise , Proteínas Sanguíneas/análise , Carboidratos/sangue , Análise Discriminante , Ácidos Graxos/sangue , Febre/sangue , Patologia Legal/métodos , Hipotermia/sangue , Lipídeos/sangue , Ácidos Nucleicos/sangue , Análise de Componente Principal , Ratos Sprague-Dawley
17.
Lab Chip ; 19(11): 1941-1952, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-30997461

RESUMO

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Centrifugação/instrumentação , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/sangue , Ácidos Nucleicos/isolamento & purificação , Automação , DNA Bacteriano/sangue , DNA Bacteriano/isolamento & purificação , Desenho de Equipamento , Escherichia coli O157/genética
18.
Nanoscale ; 11(8): 3633-3638, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30741288

RESUMO

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 µL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.


Assuntos
DNA Bacteriano/análise , Endonucleases Flap/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Chlamydia trachomatis/genética , DNA/análise , DNA/sangue , DNA/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/sangue , DNA de Cadeia Simples/metabolismo , Humanos , Limite de Detecção , Ácidos Nucleicos/sangue , Ácidos Nucleicos/metabolismo , Compostos Orgânicos/química
19.
Ann Hematol ; 97(12): 2447-2454, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30056581

RESUMO

The recently introduced Revised International Staging System (R-ISS) for multiple myeloma (MM) integrates albumin, ß2 microglobulin, lactate dehydrogenase (LDH) with high-risk cytogenetic aberrations (CA), i.e., t(4;14) and t(14;16) and del17p using fluorescent in situ hybridization (FISH). We evaluated utility of nucleic acid-based tests of multiplex ligation-based probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qRT-PCR) to define the CA and the R-ISS categories as per this approach were evaluated for their ability to predict outcome in terms of response, progression-free (PFS), and overall survival (OS). In this study (n = 180), 17 (9.4%), 118 (65.6%), and 45 (25%) patients were assigned to R-ISS1, R-ISS2, and R-ISS3 categories with statistically significant differences in median PFS (p = 0.02) and OS (p < 0.001).On univariate analysis, serum creatinine, LDH, 17p deletion, chromosome 1q gain, and response after first induction therapy were associated with statistically significant differences (p < 0.05) in PFS and in addition, age> 65 years and use of triplet therapy with OS. On multivariate analysis, only serum creatinine, LDH, and response after first induction therapy retained significance for predicting PFS and in addition, use of triplet therapy retained significance for the OS. The proposed nucleic acid-based algorithm using qRT-PCR and MLPA for R-ISS is resource-effective in terms of small quantities of sample required; feasibility of batch processing and reduced overall cost for the total number of regions evaluated and retained the prognostic significance of R-ISS, making it suitable for clinical practice for molecular characterization of MM.


Assuntos
Algoritmos , Aberrações Cromossômicas , Mieloma Múltiplo , Reação em Cadeia da Polimerase Multiplex , Ácidos Nucleicos , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Estadiamento de Neoplasias , Ácidos Nucleicos/sangue , Ácidos Nucleicos/genética
20.
Lab Chip ; 18(13): 1928-1935, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29881833

RESUMO

Integrated devices for automated nucleic acid testing (NAT) are critical for infectious disease diagnosis to be performed outside of centralized laboratories. The gold standard methods for NAT are enzymatic amplification methods like the polymerase chain reaction that typically require expensive equipment and highly-trained personnel, limiting use in low-resource settings. A low-cost, integrated, rapid, portable and user-friendly point-of-care (POC) nucleic acid diagnostic device will improve the accessibility of NAT. Here, we present a fully integrated and simple-to-use POC device operated by a passive fluidic method that is able to perform a sequential multi-step assay to detect viral nucleic acids in blood. This simple device enabled the rapid detection of hepatitis C virus in blood in approximately 30 minutes with minimal sample handling by the user.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/sangue , Desenho de Equipamento , Hepacivirus/genética , Hepatite C/diagnóstico , Humanos , Ácidos Nucleicos/sangue
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