Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 366.273
Filtrar
1.
J Clin Virol ; 146: 105061, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34973474

RESUMO

Many SARS-CoV-2 antibody detection assays have been developed but their differential performance is not well described. In this study we compared an in-house (KWTRP) ELISA which has been used extensively to estimate seroprevalence in the Kenyan population with WANTAI, an ELISA which has been approved for widespread use by the WHO. Using a wide variety of sample sets including pre-pandemic samples (negative gold standard), SARS-CoV-2 PCR positive samples (positive gold standard) and COVID-19 test samples from different periods (unknowns), we compared performance characteristics of the two assays. The overall concordance between WANTAI and KWTRP was 0.97 (95% CI, 0.95-0.98). For WANTAI and KWTRP, sensitivity was 0.95 (95% CI 0.90-0.98) and 0.93 (95% CI 0.87-0.96), respectively. Specificity for WANTAI was 0.98 (95% CI, 0.96-0.99) and 0.99 (95% CI 0.96-1.00) while KWTRP specificity was 0.99 (95% CI, 0.98-1.00) and 1.00 using pre-pandemic blood donors and pre-pandemic malaria cross-sectional survey samples respectively. Both assays show excellent characteristics to detect SARS-CoV-2 antibodies.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Antivirais , Proteínas de Transporte , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G , Quênia/epidemiologia , SARS-CoV-2 , Sensibilidade e Especificidade , Estudos Soroepidemiológicos
3.
Korean J Radiol ; 23(1): 52-59, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34983093

RESUMO

OBJECTIVE: To investigate whether the diagnostic performance of CT angiography (CTA) could be improved by modifying the conventional criterion (anastomosis site abnormality) to diagnose hepatic artery occlusion (HAO) after liver transplantation (LT) in suspected patients with Doppler ultrasound (US) abnormalities. MATERIALS AND METHODS: One hundred thirty-four adult LT recipients (88 males and 46 females; mean age, 52.7 years) with suspected HAO on Doppler US (40 HAO and 94 non-HAO according to the reference standards) were included. We evaluated 1) abnormalities in the HA anastomosis, categorized as a cutoff, ≥ 50% stenosis at the anastomotic site, or diffuse stenosis at both graft and recipient sides around the anastomosis, and 2) abnormalities in the distal run-off, including invisibility or irregular, faint, and discontinuous enhancement. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the conventional (considering anastomosis site abnormalities alone) and modified CTA criteria (abnormalities in both the anastomosis site and distal run-off) for the diagnosis of HAO were calculated and compared using the McNemar test. RESULTS: By using the conventional criterion to diagnose HAO, the sensitivity, specificity, PPV, NPV, and accuracy were 100% (40/40), 74.5% (70/94), 62.5% (40/64), 100% (70/70), and 82.1% (110/134), respectively. The modified criterion for diagnosing HAO showed significantly increased specificity (93.6%, 88/94) and accuracy (93.3%, 125/134) compared to that with the conventional criterion (p = 0.001 and 0.002, respectively), although the sensitivity (92.5%, 37/40) decreased slightly without statistical significance (p = 0.250). CONCLUSION: The modified criterion considering abnormalities in both the anastomosis site and distal run-off improved the diagnostic performance of CTA for HAO in suspected patients with Doppler US abnormalities, particularly by increasing the specificity.


Assuntos
Transplante de Fígado , Adulto , Angiografia , Angiografia por Tomografia Computadorizada , Feminino , Artéria Hepática/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Ultrassonografia Doppler
4.
Korean J Radiol ; 23(1): 139-149, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34983100

RESUMO

OBJECTIVE: To compare the effects of bone suppression imaging using deep learning (BSp-DL) based on a generative adversarial network (GAN) and bone subtraction imaging using a dual energy technique (BSt-DE) on radiologists' performance for pulmonary nodule detection on chest radiographs (CXRs). MATERIALS AND METHODS: A total of 111 adults, including 49 patients with 83 pulmonary nodules, who underwent both CXR using the dual energy technique and chest CT, were enrolled. Using CT as a reference, two independent radiologists evaluated CXR images for the presence or absence of pulmonary nodules in three reading sessions (standard CXR, BSt-DE CXR, and BSp-DL CXR). Person-wise and nodule-wise performances were assessed using receiver-operating characteristic (ROC) and alternative free-response ROC (AFROC) curve analyses, respectively. Subgroup analyses based on nodule size, location, and the presence of overlapping bones were performed. RESULTS: BSt-DE with an area under the AFROC curve (AUAFROC) of 0.996 and 0.976 for readers 1 and 2, respectively, and BSp-DL with AUAFROC of 0.981 and 0.958, respectively, showed better nodule-wise performance than standard CXR (AUAFROC of 0.907 and 0.808, respectively; p ≤ 0.005). In the person-wise analysis, BSp-DL with an area under the ROC curve (AUROC) of 0.984 and 0.931 for readers 1 and 2, respectively, showed better performance than standard CXR (AUROC of 0.915 and 0.798, respectively; p ≤ 0.011) and comparable performance to BSt-DE (AUROC of 0.988 and 0.974; p ≥ 0.064). BSt-DE and BSp-DL were superior to standard CXR for detecting nodules overlapping with bones (p < 0.017) or in the upper/middle lung zone (p < 0.017). BSt-DE was superior (p < 0.017) to BSp-DL in detecting peripheral and sub-centimeter nodules. CONCLUSION: BSp-DL (GAN-based bone suppression) showed comparable performance to BSt-DE and can improve radiologists' performance in detecting pulmonary nodules on CXRs. Nevertheless, for better delineation of small and peripheral nodules, further technical improvements are required.


Assuntos
Neoplasias Pulmonares , Nódulos Pulmonares Múltiplos , Nódulo Pulmonar Solitário , Adulto , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Curva ROC , Radiografia Torácica , Sensibilidade e Especificidade , Nódulo Pulmonar Solitário/diagnóstico por imagem
5.
Pediatr Emerg Care ; 38(1): e17-e22, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34986583

RESUMO

OBJECTIVES: The aim of the study was to determine whether point-of-care ultrasound (US) can decrease x-rays in children with ankle injuries. Secondary objectives were to determine the test performance characteristics for ankle US, analyze diagnostic errors, and compare US with the Ottawa Ankle Rules (OAR). METHODS: This was a prospective study of children younger than 21 years presenting to an emergency department with an ankle injury requiring x-rays. Pediatric emergency medicine physicians received a 1-hour training session, performed ankle US with a standardized scanning protocol of the distal tibia and fibula, and described the US as positive, negative, or equivocal for fracture. Ankle x-ray interpretation by a radiologist was the reference standard for fracture. RESULTS: One hundred twenty patients with a mean age of 13.5 (±4.0) years were enrolled. Nine patients (7.5%) had an ankle fracture on x-ray, and 56 patients (47%) had open physes. Ankle US would reduce x-rays by 81 (67.5%), missing 2 intra-articular, nondisplaced, tibial fractures in patients with open physes. Ankle US had a sensitivity of 78% (95% confidence interval [CI], 40%-97%), specificity of 71% (95% CI, 62%-79%), likelihood ratio for a positive test of 2.7 (95% CI, 1.7-4.3), and likelihood ratio for a negative test of 0.31 (95% CI, 0.09-1.07). The OAR would reduce x-rays by 21 (17.5%), missing one fracture. Ultrasound with OAR would reduce x-rays by 20 (17%) with no missed fractures. CONCLUSIONS: Point-of-care US has the potential to reduce x-rays for children with ankle injuries; however, nondisplaced, intra-articular tibial fractures may be missed. Ultrasound with OAR may reduce radiographs without missed fractures in this population.


Assuntos
Traumatismos do Tornozelo , Fraturas Ósseas , Fraturas da Tíbia , Adolescente , Traumatismos do Tornozelo/diagnóstico por imagem , Criança , Serviço Hospitalar de Emergência , Fraturas Ósseas/diagnóstico por imagem , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Estudos Prospectivos , Sensibilidade e Especificidade , Ultrassonografia
6.
PLoS One ; 17(1): e0262258, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34986156

RESUMO

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.


Assuntos
COVID-19/diagnóstico , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , COVID-19/virologia , Humanos , Influenza Humana/virologia , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Estudos Retrospectivos , Sensibilidade e Especificidade
7.
Vet Parasitol ; 301: 109641, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34979475

RESUMO

Ovine neosporosis, caused by the Apicomplexan parasite Neospora caninum, leads to reproductive failure worldwide. Nowadays, there is a trend to develop diagnostic techniques using non-invasive samples, such as milk, in order to reduce animal stress, sample collection effort, and costs. The objective of this study was to develop and validate a highly sensitive and specific serological technique, based on a time resolved-fluorescence immunoassay using a N. caninum GRA7 antigen (GRA7-TRFIA), for the detection of anti-N. caninum immunoglobulins G on sheep' full-cream milk samples. An analytical validation was performed, including intra- and inter-assay precision, analytical sensitivity and accuracy. The diagnostic performance of the assay was evaluated by studying the positive-negative discrimination by Mann Whitney U tests. In additon optimal cut-offs, diagnostic sensitivity and specificity, and areas under the curve were calculated by three Receiver Operating Curve (ROC) analyses, using GRA7-TRFIA and a N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET-ELISA) in blood sera, and the coinciding results of both techniques, as reference techniques. Moreover, Spearman's correlation of GRA7-TRFIA in milk with the techniques in sera and agreement (kappa values) were also estimated. GRA7-TRFIA for milk samples showed an adequate precision, with high analytical sensitivity and accuracy. Regarding ROC analyses, at the optimal cut-offs, the diagnostic sensitivity and specificity were more than 90 % in all cases. In addition, GRA7-TRFIA values in milk were more positively correlated to GRA7-TRFIA values in blood sera than in the case of values obtained with NcSALUVET-ELISA. GRA7-TRFIA in milk showed an almost perfect agreement with GRA7-TRFIA in blood sera (kappa = 0.98) and with the coinciding results of GRA7-TRFIA and NcSALUVET in blood sera (kappa = 1.00), while it has a substantial agreement with NcSALUVET-ELISA (kappa = 0.69). In the light of these results, GRA7-TRFIA in full-cream milk samples is a highly sensitive technique that could be used for screening anti-N. caninum antibodies in sheep flocks.


Assuntos
Doenças dos Bovinos , Coccidiose , Neospora , Doenças dos Ovinos , Animais , Anticorpos Antiprotozoários , Bovinos , Coccidiose/diagnóstico , Coccidiose/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Leite , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/diagnóstico
8.
Saudi Med J ; 43(1): 9-30, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35022280

RESUMO

OBJECTIVES: To evaluate the diagnostic utility of self-collected saliva in coronavirus desease-19 (COVID-19) screening procedures. METHODS: A total of 6 databases were reviewed from their inception until August 2021. Sensitivity and specificity were measured by extracting items (true-positive, true-negative, false-positive and false-negative) from each paper. We evaluated the diagnostic accuracy based on Quality Assessment of Diagnostic Accuracy Studies, version 2. RESULTS: A total of 41 studies were included in the final analysis. The diagnostic odds ratio (OR) of self-collected saliva was 196.2022 (95% confidence interval [CI]: 117.8833-326.5546). The area under the summary receiver operating characteristic curve was 0.955. For detecting COVID-19, self-collected saliva had a moderate sensitivity of 0.8476 [0.8045-0.8826] and positive predictive value of 0.9404 [0.9122-0.9599] but high specificity of 0.9817 [0.9707-0.9887] and negative predictive value of 0.9467 [0.9130-0.9678]. In a subgroup analysis, the diagnostic accuracy of self-collected saliva tended to be higher for symptomatic (vs. asymptomatic) examinees. CONCLUSION: Although naso/oropharyngeal swab tests are the most accurate and important diagnostic tools, the saliva-based testing method can be used as a suitable alternative test, with the advantages of increased patient convenience, efficient testing, and the need for fewer medical staff and resources. In particular, simple collecting method such as drooling or spitting without coughing would be effective in evaluating the symptomatic patients.PROSPERO no.: CRD42021279287.


Assuntos
COVID-19 , Saliva , Humanos , Nasofaringe , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Sensibilidade e Especificidade , Manejo de Espécimes
9.
Clin Nucl Med ; 47(1): 36-42, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34661556

RESUMO

PURPOSE: This study investigated diagnostic accuracies of 18F-FDG PET or PET/CT for characterization of histologic type of thymic epithelial tumors (TETs) through a systematic review and meta-analysis. PATIENTS AND METHODS: The PubMed, Cochrane database, and EMBASE database, from the earliest available date of indexing through August 31, 2020, were searched for studies evaluating diagnostic performance of 18F-FDG PET or PET/CT for characterization of TET. We determined the sensitivities and specificities, calculated positive and negative likelihood ratios (LR+ and LR-), and constructed summary receiver operating characteristic curves. RESULTS: The pooled sensitivity of 18F-FDG PET or PET/CT was 0.89 (95% confidence interval [CI], 0.80-0.95), and the pooled specificity was 0.77 (95% CI, 0.63-0.87) for differentiation between thymic cancer and thymoma. Likelihood ratio syntheses gave an overall positive likelihood ratio (LR+) of 3.9 and negative likelihood ratio (LR-) of 0.14. The pooled diagnostic odds ratio was 28 (95% CI, 13-63). The pooled sensitivity was 0.90 (95% CI, 0.75-0.96), and the pooled specificity was 0.81 (95% CI, 0.68-0.89) for differential diagnosis of a low-risk or high-risk TET. LR+ was 4.7 and LR- was 0.12. The pooled diagnostic odds ratio was 38 (95% CI, 12-121). In meta-regression analysis, no variable was the source of the study heterogeneity. CONCLUSIONS: 18F-FDG PET or PET/CT has excellent diagnostic performances for characterization of TET. Further large multicenter studies would be necessary to establish the diagnostic accuracy of 18F-FDG PET or PET/CT for differentiation of histologic type of TET.


Assuntos
Neoplasias Epiteliais e Glandulares , Neoplasias do Timo , Testes Diagnósticos de Rotina , Fluordesoxiglucose F18 , Humanos , Neoplasias Epiteliais e Glandulares/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Sensibilidade e Especificidade , Neoplasias do Timo/diagnóstico por imagem
10.
J Virol Methods ; 299: 114339, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687784

RESUMO

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.


Assuntos
COVID-19 , Ácidos Nucleicos , Teste para COVID-19 , Humanos , Indicadores e Reagentes , Fenômenos Magnéticos , Pandemias , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
11.
J Virol Methods ; 299: 114337, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687785

RESUMO

In Emergency Room, Point-of-care antigen testing for SARS-CoV-2 antigen can expedite clinical strategies for patient management. We tested 1,232 consecutive patients during Italian second wave peak using the recent LumiraDx microfluidic assay. This assay showed high concordance (96.9 %), sensitivity and specificity compared to molecular testing, being highly valuable.


Assuntos
COVID-19 , SARS-CoV-2 , Antígenos Virais , Serviço Hospitalar de Emergência , Humanos , Microfluídica , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , Sensibilidade e Especificidade
12.
Biosens Bioelectron ; 196: 113689, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34688112

RESUMO

We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5'-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.5 copies/µL (= 10 copies/reaction) for both envelope (E) and nucleocapsid (N) genes within 30 min under isothermal temperature (41 °C). When the NESBA was applied to test a large cohort of clinical samples (n = 98), the results fully agreed with those from qRT-PCR and showed the excellent accuracy by yielding 100% clinical sensitivity and specificity. By employing multiple molecular beacons with different fluorophore labels, the NESBA was further modulated to achieve multiplex molecular diagnostics, so that the E and N genes of SARS-CoV-2 gRNA were simultaneously assayed in one-pot. By offering the superior analytical performances over the current qRT-PCR, the isothermal NESBA technique could serve as very powerful platform technology to realize the point-of-care (POC) diagnosis for COVID-19.


Assuntos
Técnicas Biossensoriais , COVID-19 , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
13.
J Virol Methods ; 299: 114338, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34695479

RESUMO

BACKGROUND: Detection of SARS-CoV-2 infections relies on the use of sensitive, accurate and high throughput RT-PCR assays. OBJECTIVES: We assessed the analytical performance of the Abbott RealTime SARS-CoV-2 (RT-SARS), Alinity m SARS-CoV-2 (AlinSARS) assays and compared the clinical performance of the RT-SARS, AlinSARS, and Alinity m Resp-4-Plex (Alin4Plex) assays to the Seegene Allplex assay (Allplex) and an inhouse test (Inhouse). RESULTS: We found 100 % positive percent agreement (PPA) and 100 % negative percent agreement (NPA) comparing RT-SARS and Allplex. RT-SARS, AlinSARS and Inhouse showed 100 % NPA and 100 % PPA across all assays, except for the RdRp target of Inhouse (PPA = 84 %). Similarly, Alin4Plex and Allplex showed high agreement with specimens containing either SARS-CoV-2, influenza A, influenza B, or RSV. Detection rates of 100 % for SARS-CoV-2 at 50 copies/mL, high precision, and no cross-reactivity with non-SARS-CoV-2 respiratory pathogens were observed for RT-SARS and AlinSARS. AlinSARS detected SARS-CoV-2 in spiked throat washes and in specimens infected with SARS-CoV-2 Alpha or Beta variants. CONCLUSIONS: The newly developed RT-SARS, AlinSARS, and Alin4Plex assays proved to be useful for detecting SARS-CoV-2 RNA in clinical samples.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , RNA Viral/genética , Sensibilidade e Especificidade
14.
J Virol Methods ; 299: 114340, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34695480

RESUMO

BACKGROUND: Screening of infectious asymptomatic or pre-symptomatic individuals for SARS-CoV-2 is at present a key to controling the COVID-19 pandemic. In order to expand testing capability and limit cost, pool testing of asymtomatic individuals has been proposed, provided assay performance is not significantly affected. METHODS: Combined nose and throat (N/T) swabs collected from COVID-19 infected or non-infected individuals were tested using SAMBA II individually and in pools of four (one positive and 3 negative). The evaluation was conducted by the manufacturer and an independent NHS site. Ct cycles of individual positives and pooled positives were determined by qRT-PCR. RESULTS: In 42 pools containing a single positive sample with Ct values ranging between 17 and 36, 41 pools (97.6 %) were found positive by the SARS-CoV-2 SAMBA II test. The false-negative pool by SAMBA was also negative by both reference methods used in this evaluation.The individual positive sample in this pool was positive by SAMBA (Orf only) and by one of the reference methods (S gene only, Ct 35) but negative by the second reference method indicating that the sample itself was very low viral load. All 78 pools containing 4 negative swabs were negative (100 % specificity). DISCUSSION: The preliminary data of the evaluation indicated a high level of performance in both sensitivity and specificity of the SAMBA II assay when used to test pools of 4 patient samples. The implementation of this pooled protocol can increase throughput and reduce cost/test when the prevalence of COVID is low.


Assuntos
COVID-19 , SARS-CoV-2 , Testes Diagnósticos de Rotina , Humanos , Pandemias , Sensibilidade e Especificidade , Manejo de Espécimes
15.
Methods Mol Biol ; 2409: 157-171, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709641

RESUMO

Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30: 545-551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure "semi-nested," generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff.


Assuntos
Vírus da Dengue , Dengue , Animais , Dengue/diagnóstico , Vírus da Dengue/genética , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
16.
Methods Mol Biol ; 2409: 173-196, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709642

RESUMO

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).


Assuntos
Vírus da Dengue , Dengue , Anticorpos Antivirais , Antígenos Virais , Dengue/diagnóstico , Vírus da Dengue/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G , Imunoglobulina M , Sensibilidade e Especificidade , Proteínas não Estruturais Virais
17.
Biosens Bioelectron ; 197: 113759, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34741956

RESUMO

The current pandemic of COVID-19 caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concerns. Rapid and accurate testing of SARS-CoV-2 is urgently needed for early detection and control of the disease spread. Here, we present an RT-LAMP coupled glass nanopore digital counting method for rapid detection of SARS-CoV-2. We validated and compared two one-pot RT-LAMP assays targeting nucleocapsid (N) and envelop (E) genes. The nucleocapsid assay was adopted due to its quick time to positive and better copy number sensitivity. For qualitative positive/negative classification of a testing sample, we used the glass nanopore to digitally count the RT-LAMP amplicons and benchmarked the event rate with a threshold. Due to its intrinsic single molecule sensitivity, nanopore sensors could capture the amplification dynamics more rapidly (quick time to positive). We validated our RT-LAMP coupled glass nanopore digital counting method for SARS-CoV-2 detection by using both spiked saliva samples and COVID-19 clinical nasopharyngeal swab samples. The results obtained showed excellent agreement with the gold standard RT-PCR assay. With its integration capability, the electronic nanopore digital counting platform has significant potential to provide a rapid, sensitive, and specific point-of-care assay for SARS-CoV-2.


Assuntos
Técnicas Biossensoriais , COVID-19 , Nanoporos , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , Sensibilidade e Especificidade
18.
Biosens Bioelectron ; 197: 113736, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34741957

RESUMO

The reverse transcription-polymerase chain reaction (RT-PCR) method has been adopted worldwide to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although this method has good sensitivity and specificity, there is a need to develop a more rapid diagnostic technology, given the virus's rapid spread. However, the RT-PCR method takes a long time to diagnose SARS-CoV-2 because of the required thermocycling steps. Therefore, we developed a surface-enhanced Raman scattering (SERS)-PCR detection method using an AuNP-internalized Au nanodimple substrate (AuNDS) to shorten the diagnosis time by reducing the number of thermocycling steps needed to amplify the DNA. For the representative target markers, namely, the envelope protein (E) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2, 25 RT-PCR thermocycles are required to reach a detectable threshold value, while 15 cycles are needed for magnetic bead-based SERS-PCR when the initial DNA concentration was 1.00× 105 copies/µL. However, only 8 cycles are needed for the AuNDS-based SERS-PCR. The corresponding detectable target DNA concentrations were 3.36 × 1012, 3.28 × 109, and 2.56 × 107 copies/µL, respectively. Therefore, AuNDS-based SERS-PCR is seen as being a new molecular diagnostic platform that can shorten the time required for the thermocycling steps relative to the conventional RT-PCR.


Assuntos
Técnicas Biossensoriais , COVID-19 , Nanopartículas Metálicas , Ouro , Humanos , Reação em Cadeia da Polimerase , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Sensibilidade e Especificidade
19.
J Virol Methods ; 299: 114352, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34748815

RESUMO

A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases - as seen with the B.1.1.7 variant - can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charité RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.


Assuntos
COVID-19 , SARS-CoV-2 , Sítios de Ligação , Humanos , Mutação , RNA Viral/genética , Sensibilidade e Especificidade
20.
Spectrochim Acta A Mol Biomol Spectrosc ; 267(Pt 1): 120533, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34749108

RESUMO

One of the most important types of evidence in certain criminal investigations is traces of human blood. For a detailed investigation, blood samples must be identified and collected at the crime scene. The present study aimed to evaluate the potential of the identification of human blood in stains deposited on different types of floor tiles (five types of ceramics and four types of porcelain tiles) using a portable NIR instrument. Hierarchical models were developed by combining multivariate analysis techniques capable of identifying traces of human blood (HB), animal blood (AB) and common false positives (CFP). The spectra of the dried stains were obtained using a portable MicroNIR spectrometer (Viavi). The hierarchical models used two decision rules, the first to separate CFP and the second to discriminate HB from AB. The first decision rule, used to separate the CFP, was based on the Q-Residual criterion considering a PCA model. For the second rule, used to discriminate HB and AB, the Q-Residual criterion were tested as obtained from a PCA model, a One-Class SIMCA model, and a PLS-DA model. The best results of sensitivity and specificity, both equal to 100%, were obtained when a PLS-DA model was employed as the second decision rule. The hierarchical classification models built for these same training sets using a PCA or SIMCA model also obtained excellent sensitivity results for HB classification, with values above 94% and 78% of specificity. No CFP samples were misclassified. Hierarchical models represent a significant advance as a methodology for the identification of human blood stains at crime scenes.


Assuntos
Manchas de Sangue , Humanos , Análise Multivariada , Análise de Componente Principal , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...