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1.
Biotechnol Bioeng ; 119(3): 994-1003, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34953069

RESUMO

Transition of rapid, ready-to-use, and low-cost nucleic acid-based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling through nucleic acid isolation and detection in the field. Here we developed a simple, portable, low-cost, colorimetric, and remotely controllable platform for reliable, high-throughput, and rapid diagnosis using loop-mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low-cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady-state temperature error of the system is <1%. We performed LAMP, Colony-LAMP, and Colony polymerase chain reactions (PCRs) using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65°C and 30 min. We detected the end-point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony-LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.


Assuntos
Colorimetria , Técnicas de Amplificação de Ácido Nucleico , Escherichia coli/genética , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/genética , Sensibilidade e Especificidade
2.
BMC Infect Dis ; 22(1): 505, 2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35641907

RESUMO

BACKGROUND: Coronavirus-2019 (COVID-2019) is a novel coronavirus known as Acute Respiratory Syndrome (SARS-CoV-2). The premier standard test for SARS-CoV-2 diagnosis is a one-step RT-qPCR method, which requires specific probes and reagents. Therefore, detection on a large scale is expensive and cannot be very accurate. METHODS: A cost-effective technique based on SYBR green was evaluated in the current study. The specific primers for S and N genes were designed, then performed the cross-reactivity test with other coronavirus and respiratory viruses positive samples. Moreover, the analytical sensitivity test was carried out with 8 dilutions (1:10). Lastly, the SARS-CoV-2 clinical samples (n = 210) were tested by these two methods, and receiver operating characteristic (ROC) analysis was performed to investigate the incremental diagnostic value of each gene in the study methods. RESULTS: The two-step method detected up to 6th dilutions of the SARS-CoV-2 samples and did not show any amplification of the positive samples of other respiratory viruses. ROC analysis revealed a diagnostic ability of the two-step method for SARS-CoV-2 with an area under the ROC curve of ≥ 0.7 (P Ë‚ 0.05) and relatively high sensitivity and specificity. The combination of N and S genes increased the sensitivity up to 88%, specificity up to 86%, and area under the ROC curve up to 0.85 (95% confidence interval (95% CI) 0.72 to 0.93, P = 0.0461). CONCLUSION: Our findings indicated that the two-step method has comparable sensitivity and specificity to the one-step method. Therefore, this method can be considered a potential diagnostic method for diagnosing and monitoring COVID-19 patients. It suggests that when the one-step RT-qPCR method is not available, the two-step RT-qPCR can be used to identify SARS-CoV-2.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade
3.
BMC Infect Dis ; 22(1): 506, 2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35641936

RESUMO

BACKGROUND: Xpert MTB/Rif, a molecular test to detect tuberculosis (TB), has been proven to have high sensitivity and specificity when compared with liquid culture in clinical settings. However, little is known about its performance in community TB screening. METHODS: In Vietnam, a national TB prevalence survey was conducted in 2017. Survey participants who screened positive by chest X-ray, cough symptoms and/or recent history of tuberculosis were requested to provide at least two sputum samples that were tested for Mycobacterium tuberculosis by Xpert MTB/Rif G4 (Xpert) and BACTEC MGIT960 culture (MGIT). RESULTS: There were 4,649 eligible participants provided both samples for testing. Among them, 236 (5.1%) participants tested positive for TB by Xpert, 244 (5.3%) tested positive by MGIT and 317 tested positive by at least one test; 163 (51.4%) had discordant test results. Of the positive Xpert, 162 (68.6%) showed a low or very low bacterial load. In multivariate logistic regression comparing discordant with Xpert-MGIT concordant positive results, discordant Xpert-positive results occurred more often among participants who had low sputum bacterial load, male sex, a history of TB treatment, or night sweats. The associated factors were male sex, abnormal chest X-ray and having night sweats when the logistic model was against those with both Xpert and MGIT negative. CONCLUSIONS: We found high rates of discordance in the performance of Xpert and MGIT for community-based TB case finding. In situations where the majority of TB cases are expected to have a low bacterial load, multiple diagnostic tests and/or multiple samples are required to reach sufficient sensitivity.


Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Feminino , Humanos , Masculino , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Escarro/microbiologia , Vietnã/epidemiologia
4.
J Med Life ; 15(4): 443-447, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35646183

RESUMO

Appendectomy is still the best treatment for acute appendicitis in pediatric patients. Given the problems of early and immediate diagnosis of acute appendicitis, defining the best diagnostic protocol for this condition is of utmost importance. Different diagnostic methods, such as Lintula and appendicitis inflammatory response (AIR) scoring systems, are used for this purpose. This study aims to compare Lintula and AIR scoring systems among children with suspicion of acute appendicitis regarding their postoperative outcomes. During two years, a prospective multicentric study was carried out in the selected hospitals of Iran. Pediatric patients admitted with the diagnosis of acute appendicitis were enrolled in the study. Before decision making, each patient's score was calculated according to two appendicitis scoring systems of Lintula and AIR. The clinical outcomes and diagnosis of patients were then compared to the results of each scoring system. For those patients who were a candidate to undergo surgery, the final diagnosis of acute appendicitis was made by histopathology. Patients were divided into high and low-risk groups according to scoring systems outcomes. Among the patients with lower scoring for appendicitis, the AIR scoring system had a sensitivity and specificity of 95%, which was more promising than that of the Lintula system (19%); however, the specificity was comparable between the two models (74% vs. 83%). For patients at higher risk of acute appendicitis, although the AIR scoring systems did not provide reliable results (sen: 45% and spe: 25%), the Lintula scoring showed remarkable sensitivity (87%), accompanied by a high diagnostic accuracy (87%). AIR and Lintula scoring systems are not accurate models to predict the risk of acute appendicitis among children; therefore, they can serve as an adjacent modality for other diagnostic methods.


Assuntos
Apendicite , Doença Aguda , Apendicectomia , Apendicite/diagnóstico , Apendicite/patologia , Apendicite/cirurgia , Criança , Humanos , Estudos Prospectivos , Sensibilidade e Especificidade
5.
Front Cell Infect Microbiol ; 12: 879887, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35646725

RESUMO

Porcine parvovirus (PPV) is one of the important causes of pig reproductive diseases. The most prevalent methods for PPV authentication are the polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and quantitative real-time PCR. However, these procedures have downsides, such as the fact that they take a long time and require expensive equipment. As a result, a rapid, visible, and economical clinical diagnostic strategy to detect PPV is necessary. In this study, three pairs of crRNA primers were designed to recognize the VP2 gene, and an ERA-CRISPR/Cas12a system for PPV detection was successfully developed. The approach involved isothermal detection at 37°C, and the method can be used for visual inspection. The detection limit of the ERA-CRISPR/Cas12a system was 3.75 × 102 copies/µL, and no cross reactions with other porcine viruses were found. In view of the preceding, a rapid, visible, and low-cost nucleic acid testing approach for PPV has been developed using the ERA-CRISPR/Cas12a system.


Assuntos
Parvovirus Suíno , Doenças dos Suínos , Animais , Sistemas CRISPR-Cas , Parvovirus Suíno/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/genética
6.
Arch Microbiol ; 204(6): 355, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35648234

RESUMO

Syphilis is a sexually transmitted disease caused by the spirochaete bacterium Treponema pallidum. This study has developed a multiplex High-Resolution Melt-curve Loop-mediated isothermal amplification (multiplex HRM-LAMP) assay targeting the marker genes polA and tprL to detect T. pallidum. The multiplex HRM-LAMP assay conditions were optimized at 65 °C for 45 min. Real-time melt-curve analysis of multiplex HRM-LAMP shows two melt-curve peaks corresponding to polA and tprL with a Tm value of 80 ± 0.5 °C and 87 ± 0.5 °C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 × 10-4 ng/µL (3.79 copies/µL) of T. pallidum. The specificity was evaluated using seven different bacterial species, and the developed method was 100% specific in detecting T. pallidum. A total of 64 blood samples of T. pallidum suspected cases were used to validate the assay method. The clinical validation showed that the assay was 96.43% sensitive and 100% specific in detecting syphilis. Thus, the developed method was more rapid and sensitive than other available methods and provides a multigene-based diagnostic approach to detect T. pallidum.


Assuntos
Sífilis , Treponema pallidum , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Sífilis/diagnóstico , Treponema pallidum/genética
7.
BMC Musculoskelet Disord ; 23(1): 577, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35705930

RESUMO

BACKGROUND: The development of computer-assisted technologies to diagnose anterior cruciate ligament (ACL) injury by analyzing knee magnetic resonance images (MRI) would be beneficial, and convolutional neural network (CNN)-based deep learning approaches may offer a solution. This study aimed to evaluate the accuracy of a CNN system in diagnosing ACL ruptures by a single slice from a knee MRI and to compare the results with that of experienced human readers. METHODS: One hundred sagittal MR images from patients with and without ACL injuries, confirmed by arthroscopy, were cropped and used for the CNN training. The final decision by the CNN for intact or torn ACL was based on the probability of ACL tear on a single MRI slice. Twelve board-certified physicians reviewed the same images used by CNN. RESULTS: The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of the CNN classification was 91.0%, 86.0%, 88.5%, 87.0%, and 91.0%, respectively. The overall values of the physicians' readings were similar, but the specificity was lower than the CNN classification for some of the physicians, thus resulting in lower accuracy for the human readers. CONCLUSIONS: The trained CNN automatically detected the ACL tears with acceptable accuracy comparable to that of human readers.


Assuntos
Lesões do Ligamento Cruzado Anterior , Traumatismos do Joelho , Ligamento Cruzado Anterior , Lesões do Ligamento Cruzado Anterior/diagnóstico por imagem , Artroscopia , Humanos , Traumatismos do Joelho/diagnóstico , Imageamento por Ressonância Magnética/métodos , Redes Neurais de Computação , Estudos Retrospectivos , Sensibilidade e Especificidade
8.
J Biomed Opt ; 27(6)2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35701871

RESUMO

SIGNIFICANCE: Accurate early diagnosis of malignant skin lesions is critical in providing adequate and timely treatment; unfortunately, initial clinical evaluation of similar-looking benign and malignant skin lesions can result in missed diagnosis of malignant lesions and unnecessary biopsy of benign ones. AIM: To develop and validate a label-free and objective image-guided strategy for the clinical evaluation of suspicious pigmented skin lesions based on multispectral autofluorescence lifetime imaging (maFLIM) dermoscopy. APPROACH: We tested the hypothesis that maFLIM-derived autofluorescence global features can be used in machine-learning (ML) models to discriminate malignant from benign pigmented skin lesions. Clinical widefield maFLIM dermoscopy imaging of 41 benign and 19 malignant pigmented skin lesions from 30 patients were acquired prior to tissue biopsy sampling. Three different pools of global image-level maFLIM features were extracted: multispectral intensity, time-domain biexponential, and frequency-domain phasor features. The classification potential of each feature pool to discriminate benign versus malignant pigmented skin lesions was evaluated by training quadratic discriminant analysis (QDA) classification models and applying a leave-one-patient-out cross-validation strategy. RESULTS: Classification performance estimates obtained after unbiased feature selection were as follows: 68% sensitivity and 80% specificity with the phasor feature pool, 84% sensitivity, and 71% specificity with the biexponential feature pool, and 84% sensitivity and 32% specificity with the intensity feature pool. Ensemble combinations of QDA models trained with phasor and biexponential features yielded sensitivity of 84% and specificity of 90%, outperforming all other models considered. CONCLUSIONS: Simple classification ML models based on time-resolved (biexponential and phasor) autofluorescence global features extracted from maFLIM dermoscopy images have the potential to provide objective discrimination of malignant from benign pigmented lesions. ML-assisted maFLIM dermoscopy could potentially assist with the clinical evaluation of suspicious lesions and the identification of those patients benefiting the most from biopsy examination.


Assuntos
Melanoma , Neoplasias Cutâneas , Dermoscopia/métodos , Humanos , Aprendizado de Máquina , Melanoma/patologia , Sensibilidade e Especificidade , Neoplasias Cutâneas/patologia
10.
Talanta ; 248: 123594, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35653961

RESUMO

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Recombinases , SARS-CoV-2/genética , Sensibilidade e Especificidade
11.
Talanta ; 248: 123644, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35660994

RESUMO

Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies/µL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Corantes Fluorescentes , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade
12.
Talanta ; 248: 123624, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35660998

RESUMO

Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many highly sensitive immunoassays, magnetic beads are used to capture fluorescently labeled target molecules. The target molecules are then quantified by detecting the fluorescent signal from individual beads, which is time consuming and requires a complicated and expensive detection system. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system, which uses a small permanent magnet to aggregate the beads into a small detection volume and eliminates background noise by steering a laser beam in and out of the cluster of beads. Shortening the aggregation, acquisition, and well-to-well scanning transition times enables reading a 96-well plate within 10 min. Using the ht-OMB system to detect human Interleukin-8, we demonstrated a limit of detection of 0.14 ng/L and a 4-log dynamic range. Testing 94 RNA extracts from 36 confirmed RT-qPCR SARS-CoV-2-positive patients (Ct≤40) and 58 confirmed RT-qPCR SARS-CoV-2-negative individuals resulted in 100% sensitivity and 100% specificity.


Assuntos
COVID-19 , SARS-CoV-2 , Biomarcadores , Humanos , Imunoensaio/métodos , RNA Viral/análise , Sensibilidade e Especificidade
13.
Talanta ; 248: 123579, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35660999

RESUMO

Lateral flow devices (LFDs) or lateral flow tests (LFTs) are one of the most widely used biosensor platforms for point-of-care (POC) diagnostics. The basic LFD design has remained largely unchanged since its first appearance, and this has limited LFD use in clinical applications due to a general lack of analytical sensitivity. We report here a comprehensive study of the use of laser-patterned geometric control barriers that influence the flow dynamics within an LFD, with the specific aim of enhancing LFD sensitivity and lowering the limit of detection (LOD). This control of sample flow produces an increase in the time available for optimizing the binding kinetics of the implemented assay. The geometric modification to the flow path is in the form of a constriction that is produced by depositing a photo-sensitive polymer onto the nitrocellulose membrane which when polymerized, creates impermeable barrier walls through the depth of the membrane. Both the position of the constriction within the flow path and the number of constrictions allow for an increase in the sensitivity because of a slower overall flow rate within the test and a larger volume of sample per unit width of the test line. For these high sensitivity LFDs (HS-LFD), through optimization of the constriction position and addition of a second constriction we attained a 62% increase in test line color intensity for the detection of procalcitonin (PCT) and were also able to lower the LOD from 10 ng/mL to 1 ng/mL. In addition, of relevance for future commercial exploitation, this also significantly decreases the antibody consumption per device leading to reduced costs for test production. We have further tested our HS-LFD with contrived human samples, validating its application for future clinical use.


Assuntos
Técnicas Biossensoriais , Colódio , Humanos , Técnicas de Amplificação de Ácido Nucleico , Polimerização , Sensibilidade e Especificidade
14.
JCO Clin Cancer Inform ; 6: e2100192, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35671415

RESUMO

PURPOSE: Early detection of ovarian cancer, the deadliest gynecologic cancer, is crucial for reducing mortality. Current noninvasive risk assessment measures include protein biomarkers in combination with other clinical factors, which vary in their accuracy. Machine learning can be applied to optimizing the combination of these features, leading to more accurate assessment of malignancy. However, the low prevalence of the disease can make rigorous validation of these tests challenging and can result in unbalanced performance. METHODS: MIA3G is a deep feedforward neural network for ovarian cancer risk assessment, using seven protein biomarkers along with age and menopausal status as input features. The algorithm was developed on a heterogenous data set of 1,067 serum specimens from women with adnexal masses (prevalence = 31.8%). It was subsequently validated on a cohort almost twice that size (N = 2,000). RESULTS: In the analytical validation data set (prevalence = 4.9%), MIA3G demonstrated a sensitivity of 89.8% and a specificity of 84.02%. The positive predictive value was 22.45%, and the negative predictive value was 99.38%. When stratified by cancer type and stage, MIA3G achieved sensitivities of 94.94% for epithelial ovarian cancer, 76.92% for early-stage cancer, and 98.04% for late-stage cancer. CONCLUSION: The balanced performance of MIA3G leads to a high sensitivity and high specificity, a combination that may be clinically useful for providers in evaluating the appropriate management strategy for their patients. Limitations of this work include the largely retrospective nature of the data set and the unequal, albeit random, assignment of histologic subtypes between the training and validation data sets. Future directions may include the addition of new biomarkers or other modalities to strengthen the performance of the algorithm.


Assuntos
Neoplasias Ovarianas , Algoritmos , Biomarcadores , Carcinoma Epitelial do Ovário , Feminino , Humanos , Redes Neurais de Computação , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/epidemiologia , Estudos Retrospectivos , Sensibilidade e Especificidade
15.
New Microbiol ; 45(2): 111-114, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35699559

RESUMO

This study aimed to validate the agreement between human papillomavirus (HPV) tests self-collected samples versus clinician cervical specimens, and the pre-analytical stability of self-sampling. One hundred and fifty-seven women aged between 25 and 65 years who presented to the gynaecological department of the "CLEMENTVILLE" clinic in Montpellier voluntarily participated in HPV screening by self-sampling. Polymerase chain reaction was used to detect the presence of HPV16, HPV18 and a pool of 12 other HPV types on the Roche Cobas 8800 System. Median age was 40 years (range 20-73 and IQR 31-49 years). The overall HPV prevalence on the population studied was 27%. The agreement between clinician cervical samples and self-collected vaginal presented good agreement (Kappa =0.90) and high sensitivity (0.91) and specificity (0.98). For swabs stored for 7 days at room temperature, the HPV results presented substantial agreement (Kappa =0.89) and high sensitivity (0.97) and specificity (0.93). Our data showed that the HPV assay performed in the self-collected vaginal samples have high consistency of results with the clinician cervical samples. The use of self-collected cervical sample could be a simple and inexpensive approach in cervical cancer screening programs due to their high pre-analytical stability.


Assuntos
Infecções por Papillomavirus , Neoplasias do Colo do Útero , Adulto , Idoso , Detecção Precoce de Câncer/métodos , Feminino , Papillomavirus Humano 16 , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal/métodos , Adulto Jovem
16.
J Vector Borne Dis ; 59(1): 29-36, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35708401

RESUMO

Diagnosis of malaria is a prominent challenge due to the endemic nature of infection. Malaria poses a great threat to global public health. The disease can be diagnosed by several techniques out of which microscopy is a known gold standard. High sensitivity of molecular techniques is making them more reliable and popular as tools for diagnosis of malaria. However, new methods are required which can fulfill the criteria of being Point of Care Test (POCT) as defined by WHO. Loop-mediated isothermal amplification (LAMP) technique amplifies DNA in an isothermal condition, and surpasses the disadvantages of conventional molecular techniques such as polymerase chain reaction. Multiplex LAMP, a modification of LAMP may emerge as a new POC for malaria diagnosis. This review deals with the use of LAMP and multiplex LAMP in diagnosis of malaria and its prospective use as point of care techniques.


Assuntos
Malária , Sistemas Automatizados de Assistência Junto ao Leito , Humanos , Malária/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos Prospectivos , Sensibilidade e Especificidade
17.
J Vector Borne Dis ; 59(1): 57-62, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35708405

RESUMO

BACKGROUND & OBJECTIVES: Microscopy is considered as the gold standard for malaria diagnosis, however sub-microscopic infections can only be detected by Polymerase chain reaction, which demands high cost and elaborate laboratory setup. The Micro-chip PCR based Truenat Malaria Pv-Pf and Pf assay is a portable solution for detection of sub-microscopic/asymptomatic cases of malaria in the field, three lots of which were evaluated for P. falciparum and P. vivax malaria. METHODS: Three lots of Truenat® Malaria Pv-Pf and Pf assay (kits) were assessed using blood samples of P. vivax and P. falciparum as well as malaria negative blood samples. DNA was extracted from the blood samples using the Trueprep Auto v2 Universal Cartridge based sample prep device and real time qPCR was performed using Truelab DUO micro PCR Analyzer with three lots of Truenat® Malaria Pv-Pf and Pf Assays. Mean, Standard deviation and one-way analysis of variance (ANOVA) was used to assess the significance of inter-lot variability in Cycle threshold values. RESULTS: The Truenat® Malaria Pv-Pf and Pf assays identified the malaria parasites with 100% accuracy. Based on the test for variance (ANOVA) the inter-lot variability in cycle threshold values were not significant, indicating a high degree of precision. INTERPRETATION & CONCLUSION: Based on high accuracy and precision between different lots, the Truenat® Malaria Pv-Pf and Pf assays were found to be suitable for the diagnosis of sub-microscopic infections in field conditions to provide support in elimination of malaria.


Assuntos
Malária Falciparum , Malária Vivax , Malária , Humanos , Malária/parasitologia , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Malária Vivax/diagnóstico , Malária Vivax/parasitologia , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
18.
PLoS One ; 17(6): e0269997, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35709075

RESUMO

A rapid and accurate diagnosis is a crucial strategy for containing the coronavirus disease (COVID-19) pandemic. Considering the obstacles to upscaling the use of RT-qPCR, rapid tests based on antigen detection (Ag-RDT) have become an alternative to enhance mass testing, reducing the time for a prompt diagnosis and virus spreading. However, the performances of several commercially available Ag-RDTs have not yet been evaluated in several countries. Here, we evaluate the performance of eight Ag-RDTs available in Brazil to diagnose COVID-19. Patients admitted to tertiary hospitals with moderate or mild COVID-19 symptoms and presenting risk factors for severe disease were included. The tests were performed using a masked protocol, strictly following the manufacturer's recommendations and were compared with RT-qPCR. The overall sensitivity of the tests ranged from 9.8 to 81.1%, and specificity greater than 83% was observed for all the evaluated tests. Overall, slight or fair agreement was observed between Ag-RDTs and RT-PCR, except for the Ag-RDT COVID-19 (Acro Biotech), in which moderate agreement was observed. Lower sensitivity of Ag-RDTs was observed for patients with cycle threshold > 25, indicating that the sensitivity was directly affected by viral load, whereas the effect of the disease duration was unclear. Despite the lower sensitivity of Ag-RDTs compared with RT-qPCR, its easy fulfillment and promptness still justify its use, even at hospital admission. However, the main advantage of Ag-RDTs seems to be the possibility of increasing access to the diagnosis of COVID-19 in patients with a high viral load, allowing immediate clinical management and reduction of infectivity and community transmission.


Assuntos
COVID-19 , Antígenos Virais/análise , Brasil/epidemiologia , COVID-19/diagnóstico , COVID-19/epidemiologia , Teste para COVID-19 , Humanos , Pandemias , Sensibilidade e Especificidade
19.
PLoS One ; 17(6): e0270060, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35709204

RESUMO

BACKGROUND: An ideal test for COVID-19 would combine the sensitivity of laboratory-based PCR with the speed and ease of use of point-of-care (POC) or home-based rapid antigen testing. We evaluated clinical performance of the Diagnostic Analyzer for Selective Hybridization (DASH) SARS-CoV-2 POC rapid PCR test. METHODS: We conducted a cross-sectional study of adults with and without symptoms of COVID-19 at four clinical sites where we collected two bilateral anterior nasal swabs and information on COVID-19 symptoms, vaccination, and exposure. One swab was tested with the DASH SARS-CoV-2 POC PCR and the second in a central laboratory using Cepheid Xpert Xpress SARS-CoV-2 PCR. We assessed test concordance and calculated sensitivity, specificity, negative and positive predictive values using Xpert as the "gold standard". RESULTS: We enrolled 315 and analyzed 313 participants with median age 42 years; 65% were female, 62% symptomatic, 75% had received ≥2 doses of mRNA COVID-19 vaccine, and 16% currently SARS-CoV-2 positive. There were concordant results for 307 tests indicating an overall agreement for DASH of 0.98 [95% CI 0.96, 0.99] compared to Xpert. DASH performed at 0.96 [95% CI 0.86, 1.00] sensitivity and 0.98 [95% CI 0.96, 1.00] specificity, with a positive predictive value of 0.85 [95% CI 0.73, 0.96] and negative predictive value of 0.996 [95% CI 0.99, 1.00]. The six discordant tests between DASH and Xpert all had high Ct values (>30) on the respective positive assay. DASH and Xpert Ct values were highly correlated (R = 0.89 [95% CI 0.81, 0.94]). CONCLUSIONS: DASH POC SARS-CoV-2 PCR was accurate, easy to use, and provided fast results (approximately 15 minutes) in real-life clinical settings with an overall performance similar to an EUA-approved laboratory-based PCR.


Assuntos
COVID-19 , Adulto , COVID-19/diagnóstico , Teste para COVID-19 , Vacinas contra COVID-19 , Técnicas de Laboratório Clínico/métodos , Estudos Transversais , Feminino , Humanos , Masculino , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase , SARS-CoV-2/genética , Sensibilidade e Especificidade
20.
Sci Rep ; 12(1): 10124, 2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35710721

RESUMO

Hepatitis C virus (HCV) infection remains a global health problem, detected only in the early stages by molecular tests. Molecular tests detect HCV RNA, which is very prone to degradation by ribonucleases, reason why blood samples must be transported and stored at - 20 °C, or even - 70 °C for long-term storage. Flinders Technology Associates (FTA) cards are a useful sampling collecting device for dry blood spot (DBS) storage, especially for low and middle-income countries (LMIC). In this study, we analyzed viral HCV RNA integrity for long-term storage at room temperature compared to - 20 °C using two different types of cards for DBS: FTA Classic and 903 Protein Saver cards. For this purpose, DBS were prepared on these cards using blood or plasma samples from HCV infected patients, and samples were analysed by conventional RT-PCR. Our results showed that 903 Protein Saver cards are the best and cheapest alternative for DBS storage at room temperature. In these conditions, we found that HCV RNA integrity lasted for up to 9 months.


Assuntos
Hepatite C , RNA Viral , Teste em Amostras de Sangue Seco/métodos , Hepacivirus/genética , Humanos , RNA Viral/análise , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Temperatura
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