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1.
PLoS One ; 16(1): e0243712, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33428641

RESUMO

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.


Assuntos
/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Testes Imediatos , RNA Viral/genética , /genética , /economia , Desenho de Equipamento , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Testes Imediatos/economia , RNA Viral/isolamento & purificação , Fatores de Tempo
2.
Trials ; 22(1): 39, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33419461

RESUMO

OBJECTIVES: In this cluster-randomised controlled study (CoV-Surv Study), four different "active" SARS-CoV-2 testing strategies for general population surveillance are evaluated for their effectiveness in determining and predicting the prevalence of SARS-CoV-2 infections in a given population. In addition, the costs and cost-effectiveness of the four surveillance strategies will be assessed. Further, this trial is supplemented by a qualitative component to determine the acceptability of each strategy. Findings will inform the choice of the most effective, acceptable and affordable strategy for SARS-CoV-2 surveillance, with the most effective and cost-effective strategy becoming part of the local public health department's current routine health surveillance activities. Investigating its everyday performance will allow us to examine the strategy's applicability to real time prevalence prediction and the usefulness of the resulting information for local policy makers to implement countermeasures that effectively prevent future nationwide lockdowns. The authors would like to emphasize the importance and relevance of this study and its expected findings in the context of population-based disease surveillance, especially in respect to the current SARS-CoV-2 pandemic. In Germany, but also in many other countries, COVID-19 surveillance has so far largely relied on passive surveillance strategies that identify individuals with clinical symptoms, monitor those cases who then tested positive for the virus, followed by tracing of individuals in close contact to those positive cases. To achieve higher effectiveness in population surveillance and to reliably predict the course of an outbreak, screening and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. To better control and manage the SARS-CoV-2 pandemic, current strategies therefore need to be complemented by an active surveillance of the wider population, i.e. routinely conducted testing and monitoring activities to identify and isolate infected individuals regardless of their clinical symptoms. Such active surveillance strategies will enable more effective prevention of the spread of the virus as they can generate more precise population-based parameters during a pandemic. This essential information will be required in order to determine the best strategic and targeted short-term countermeasures to limit infection spread locally. TRIAL DESIGN: This trial implements a cluster-randomised, two-factorial controlled, prospective, interventional, single-blinded design with four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy. PARTICIPANTS: Eligible are individuals age 7 years or older living in Germany's Rhein-Neckar Region who consent to provide a saliva sample (all four arms) after completion of a brief questionnaire (two arms only). For the qualitative component, different samples of study participants and non-participants (i.e. eligible for study, but refuse to participate) will be identified for additional interviews. For these interviews, only individuals age 18 years or older are eligible. INTERVENTION AND COMPARATOR: Of the four surveillance strategies to be assessed and compared, Strategy A1 is considered the gold standard for prevalence estimation and used to determine bias in other arms. To determine the cost-effectiveness, each strategy is compared to status quo, defined as the currently practiced passive surveillance approach. Strategy A1: Individuals (one per household) receive information and study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the sample is tested for SARS-CoV-2 using Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP). Strategy A2: Individuals (one per household) receive information and study material by mail with instructions on how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. Strategy B1: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms and risk exposures. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the saliva sample is tested for SARS-CoV-2 using RT-LAMP. Strategy B2: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. In each strategy, RT-LAMP positive samples are additionally analyzed with qPCR in order to minimize the number of false positives. MAIN OUTCOMES: The identification of the one best strategy will be determined by a set of parameters. Primary outcomes include costs per correctly screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Secondary outcomes include participation rate, costs per asymptomatic case, prevalence estimates, number of asymptomatic cases per study arm, ratio of symptomatic to asymptomatic cases per study arm, participant satisfaction. Additional study components (not part of the trial) include cost effectiveness of each of the four surveillance strategies compared to passive monitoring (i.e. status quo), development of a prognostic model to predict hospital utilization caused by SARS-CoV-2, time from test shipment to test application and time from test shipment to test result, and perception and preferences of the persons to be tested with regard to test strategies. RANDOMISATION: Samples are drawn in three batches of three continuous weeks. Randomisation follows a two-stage process. First, a total of 220 sampling points have been allocated to the three different batches. To obtain an integer solution, the Cox-algorithm for controlled rounding has been used. Afterwards, sample points have been drawn separately per batch, following a probability proportional to size (PPS) random sample. Second, for each cluster the same number of residential addresses is randomly sampled from the municipal registries (self-weighted sample of individuals). The 28,125 addresses drawn per municipality are then randomly allocated to the four study arms A1, A2, B1, and B2 in the ratio 5 to 2.5 to 14 to 7 based on the expected response rates in each arm and the sensitivity and specificity of the pre-screening tool as applied in strategy B1 and B2. Based on the assumptions, this allocation should yield 2500 saliva samples in each strategy. Although a municipality can be sampled by multiple batches and the overall number of addresses per municipality might vary, the number of addresses contacted in each arm is kept constant. BLINDING (MASKING): The design is single-blinded, meaning the staff conducting the SARS-CoV-2 tests are unaware of the study arm assignment of each single participant and test sample. SAMPLE SIZES: Total sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (i.e. 2,500 participants per arm). For the qualitative component, up to 60 in-depth interviews will be conducted with about 30 study participants (up to 15 in each arm A and B) and 30 participation refusers (up to 15 in each arm A and B) purposefully selected from the quantitative study sample to represent a variety of gender and ages to explore experiences with admission or rejection of study participation. Up to 25 asymptomatic SARS-CoV-2 positive study participants will be purposefully selected to explore the way in which asymptomatic men and women diagnosed with SARS-CoV-2 give meaning to their diagnosis and to the dialectic between feeling concurrently healthy and yet also being at risk for transmitting COVID-19. In addition, 100 randomly selected study participants will be included to explore participants' perspective on testing processes and implementation. TRIAL STATUS: Final protocol version is "Surveillance_Studienprotokoll_03Nov2020_v1_2" from November 3, 2020. Recruitment started November 18, 2020 and is expected to end by or before December 31, 2020. TRIAL REGISTRATION: The trial is currently being registered with the German Clinical Trials Register (Deutsches Register Klinischer Studien), DRKS00023271 ( https://www.drks.de/drks_web/navigate.do?navigationId=trial . HTML&TRIAL_ID=DRKS00023271). Retrospectively registered 30 November 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.


Assuntos
/economia , /economia , Custos de Cuidados de Saúde , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Saliva/virologia , Inquéritos e Questionários/economia , /epidemiologia , Análise Custo-Benefício , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Vigilância da População , Valor Preditivo dos Testes , Prevalência , Ensaios Clínicos Controlados Aleatórios como Assunto , Reprodutibilidade dos Testes , Método Simples-Cego
3.
Biosens Bioelectron ; 177: 113005, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33486135

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has been a major public health challenge in 2020. Early diagnosis of COVID-19 is the most effective method to control disease spread and prevent further mortality. As such, a high-precision and rapid yet economic assay method is urgently required. Herein, we propose an innovative method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using isothermal amplification of nucleic acids on a mesh containing multiple microfluidic pores. Hybridization of pathogen DNA and immobilized probes forms a DNA hydrogel by rolling circle amplification and, consequently, blocks the pores to prevent fluid movement, as observed. Following optimization of several factors, including pore size, mesh location, and precision microfluidics, the limit of detection (LOD) for SARS-CoV-2 was determined to be 0.7 aM at 15-min incubation. These results indicate rapid, easy, and effective detection with a moderate-sized LOD of the target pathogen by remote point-of-care testing and without the requirement of any sophisticated device.


Assuntos
/métodos , Hidrogéis/química , Ácidos Nucleicos Imobilizados/química , Testes Imediatos , /isolamento & purificação , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , /economia , Sondas de DNA/química , Sondas de DNA/genética , Desenho de Equipamento , Humanos , Ácidos Nucleicos Imobilizados/genética , Dispositivos Lab-On-A-Chip , Limite de Detecção , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , /genética
4.
BMC Infect Dis ; 20(1): 783, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-33081710

RESUMO

BACKGROUND: A cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting. METHODS: This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. RESULTS: The novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a "gold standard", the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. CONCLUSIONS: RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/métodos , Dados Preliminares , Idoso , Idoso de 80 Anos ou mais , Infecções por Coronavirus/virologia , Confiabilidade dos Dados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/economia , Estudos Prospectivos , Sensibilidade e Especificidade
5.
Nat Protoc ; 15(11): 3663-3677, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33005038

RESUMO

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.


Assuntos
Celulose/química , Ácidos Nucleicos/isolamento & purificação , Animais , Bactérias/química , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Plantas/química , Fatores de Tempo , Vírus/química
6.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32900935

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Colorimetria , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Poliproteínas , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas Virais/genética , Inativação de Vírus
7.
PLoS One ; 15(8): e0237187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833981

RESUMO

INTRODUCTION: Infection of equids with Trypanosoma brucei (T. brucei) ssp. is of socioeconomic importance across sub-Saharan Africa as the disease often progresses to cause fatal meningoencephalitis. Loop-mediated isothermal amplification (LAMP) has been developed as a cost-effective molecular diagnostic test and is potentially applicable for use in field-based laboratories. PART I: Threshold levels for T. brucei ssp. detection by LAMP were determined using whole equine blood specimens spiked with known concentrations of parasites. Results were compared to OIE antemortem gold standard of T. brucei-PCR (TBR-PCR). RESULTS I: Threshold for detection of T. brucei ssp. on extracted DNA from whole blood was 1 parasite/ml blood for LAMP and TBR-PCR, and there was excellent agreement (14/15) between tests at high (1 x 103/ml) concentrations of parasites. Detection threshold was 100 parasites/ml using LAMP on whole blood (LWB). Threshold for LWB improved to 10 parasites/ml with detergent included. Performance was excellent for LAMP at high (1 x 103/ml) concentrations of parasites (15/15, 100%) but was variable at lower concentrations. Agreement between tests was weak to moderate, with the highest for TBR-PCR and LAMP on DNA extracted from whole blood (Cohen's kappa 0.95, 95% CI 0.64-1.00). PART II: A prospective cross-sectional study of working equids meeting clinical criteria for trypanosomiasis was undertaken in The Gambia. LAMP was evaluated against subsequent TBR-PCR. RESULTS II: Whole blood samples from 321 equids in The Gambia were processed under field conditions. There was weak agreement between LWB and TBR-PCR (Cohen's kappa 0.34, 95% CI 0.19-0.49) but excellent agreement when testing CSF (100% agreement on 6 samples). CONCLUSIONS: Findings support that LAMP is comparable to PCR when used on CSF samples in the field, an important tool for clinical decision making. Results suggest repeatability is low in animals with low parasitaemia. Negative samples should be interpreted in the context of clinical presentation.


Assuntos
Doenças dos Cavalos/parasitologia , Cavalos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/diagnóstico , Tripanossomíase Africana/veterinária , Animais , Estudos Transversais , DNA de Protozoário/sangue , DNA de Protozoário/genética , Feminino , Gâmbia , Masculino , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Reação em Cadeia da Polimerase/economia , Estudos Prospectivos , Sensibilidade e Especificidade , Tripanossomíase Africana/parasitologia
8.
PLoS Pathog ; 16(8): e1008705, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853291

RESUMO

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/genética , Pneumonia Viral/diagnóstico , Pneumonia Viral/genética , Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genes Virais/genética , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
9.
PLoS One ; 15(8): e0237655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810167

RESUMO

BACKGROUND: Several tests are available for plague confirmation but bacteriological culture with Yersinia pestis strain isolation remains the gold standard according to the World Health Organization. However, this is a time consuming procedure; requiring specific devices and well-qualified staff. In addition, strain isolation is challenging if antibiotics have been administered prior to sampling. Here, we developed a loop-mediated isothermal amplification (LAMP) technique, a rapid, simple, sensitive and specific technique that would be able to detect Y. pestis in human biological samples. METHODS: LAMP primers were designed to target the caf1 gene which is specific to Y. pestis. The detection limit was determined by testing 10-fold serial dilution of Y. pestis DNA. Cross-reactivity was tested using DNA extracts from 14 pathogens and 47 residual samples from patients suffering from non-plague diseases. Specificity and sensitivity of the LAMP caf1 were assessed on DNA extracts of 160 human biological samples. Then, the performance of the LAMP caf1 assay was compared to conventional PCR and bacteriological culture. RESULTS: The detection limit of the developed Y. pestis LAMP assay was 3.79 pg/µl, similar to conventional PCR. The result could be read out within 45 min and as early as 35 minutes in presence of loop primer, using a simple water bath at 63°C. This is superior to culture with respect to time (requires up to 10 days) and simplicity of equipment compared to PCR. Furthermore, no cross-reactivity was found when tested on DNA extracts from other pathogens and human biological samples from patients with non-plague diseases. Compared to the gold standard, LAMP sensitivity and specificity were 97.9% (95% CI: 89.1%-99.9%) and 94.6% (95% CI: 88.6%-97.9%), respectively. CONCLUSION: LAMP detected Y. pestis effectively with high sensitivity and specificity in human plague biological samples. It can potentially be used in the field during outbreaks in resource limited countries such as Madagascar.


Assuntos
Técnicas Bacteriológicas/métodos , DNA Bacteriano/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Peste/diagnóstico , Yersinia pestis/isolamento & purificação , Técnicas Bacteriológicas/economia , Estudos de Viabilidade , Humanos , Limite de Detecção , Madagáscar , Técnicas de Amplificação de Ácido Nucleico/economia , Peste/microbiologia , Fatores de Tempo , Yersinia pestis/genética
10.
BMC Infect Dis ; 20(1): 525, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32689953

RESUMO

BACKGROUND: Neisseria meningitidis is a major cause of bacterial meningitis, and these infections are associated with a high mortality rate. Rapid and reliable diagnosis of bacterial meningitis is critical in clinical practice. However, this disease often occurs in economically depressed areas, so an inexpensive, easy to use, and accurate technology is needed. We performed a pooled-analysis to assess the potential of the recently developed loop-mediated isothermal amplification (LAMP) assay for detection of meningococcus. METHODS: Pubmed, Embase, and Web of Science were searched to identify original studies that used the LAMP assay to detect meningococcus. After pooling of data, the sensitivity and specificity were calculated, a summary receiver operating characteristic (SROC) curve was determined, and the area under the SROC curve was computed to determine diagnostic accuracy. Publication bias was assessed using Deek's funnel plot. RESULTS: We examined 14 studies within 6 publications. The LAMP assay had high sensitivity (94%) and specificity (100%) in the detection of meningococcus in all studies. The area under the SROC curve (0.980) indicated high overall accuracy of the LAMP assay. There was no evidence of publication bias. DISCUSSION: The LAMP assay has accuracy comparable to bacterial culture and PCR for detection of meningococcus, but is less expensive and easier to use. We suggest the adoption of the LAMP assay to detect meningococcus, especially in economically depressed areas.


Assuntos
Meningite Meningocócica/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Neisseria meningitidis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Confiabilidade dos Dados , Humanos , Meningite Meningocócica/microbiologia , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Curva ROC , Sensibilidade e Especificidade
11.
PLoS One ; 15(6): e0234682, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530929

RESUMO

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Testes Imediatos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Primers do DNA , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Sensibilidade e Especificidade , Fatores de Tempo
12.
Med Hypotheses ; 141: 109786, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32361529

RESUMO

Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Primers do DNA , Diagnóstico Precoce , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias/economia , Pneumonia Viral/economia , Pneumonia Viral/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/economia , Sensibilidade e Especificidade , Fatores de Tempo
13.
Emerg Microbes Infect ; 9(1): 998-1007, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32306853

RESUMO

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/normas , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Fatores de Tempo
14.
Int J Infect Dis ; 95: 98-105, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32278935

RESUMO

OBJECTIVES: To report the cost-effectiveness of Xpert in detecting Mycobacterium tuberculosis (MTB) through a comprehensive systematic review. METHODS: Specialized bibliographic databases were searched. Study quality was evaluated by commonly-used industry standards. Due to heterogeneity, evidences were synthesized narratively. RESULTS: Four studies from intermediate-to-low tuberculosis (TB)-burdern areas and 17 studies from high-TB-burden areas were included. Smear microscopy, clinical diagnosis and chest radiography were mostly used for comparison. Cost elements varied considerably depending on the perspectives. Cost-effectiveness and cost-utility analyses were used by seven and fourteen studies, respectively. All studies were of high quality (CHEERS score of 78.4 and QHES score of 86.9). Average cost per test was 29.8 US$ for Xpert compared with 3.83 US$ for smear microscopy. Cost-effectiveness analyses mostly supported application of Xpert into areas under varying TB burdens. CONCLUSIONS: Xpert seems cost-effective under respective willingness-to-pay thresholds in nations with differences in socioeconomy, HIV stress and geographical distribution. Nevertheless, policymakers will benefit from localized studies since regional economic/financial statuses and health-care system should also be considered apart from the reports of cost-effectiveness.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/economia , Tuberculose/microbiologia , Análise Custo-Benefício , Humanos , Tuberculose/diagnóstico , Tuberculose/economia
15.
Dis Markers ; 2020: 8074314, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184904

RESUMO

Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi which is endemic throughout Latin America and is spread by worldwide migration. Diagnosis is currently limited to serological and molecular techniques having variations regarding their sensitivity and specificity. This work was aimed at developing a new sensitive, applicable, and cost-effective molecular diagnosis technique for loop-mediated isothermal amplification-based detection of T. cruzi (Tc-LAMP). The results led to determining a highly homologous satellite repeat region (231 bp) among parasite strains as a molecular marker for diagnosing the disease. Tc-LAMP was performed correctly for detecting parasite DNA (5 fg for the CL Brener strain and 50 fg for the DM28, TcVI, and TcI strains). Assay results proved negative for DNA from 16 helminth species and 7 protozoa, including Leishmania spp. Tc-LAMP based on the highly repeated T. cruzi satellite region is thus proposed as an important alternative for diagnosing T. cruzi infection, overcoming other methods' limitations such as their analytic capability, speed, and requiring specialized equipment or highly trained personnel. Tc-LAMP could be easily adapted for point-of-care testing in areas having limited resources.


Assuntos
Doença de Chagas/diagnóstico , DNA Satélite/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Trypanosoma cruzi/isolamento & purificação , DNA de Protozoário/genética , Marcadores Genéticos , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Testes Imediatos , Sensibilidade e Especificidade , Trypanosoma cruzi/genética
16.
BMC Infect Dis ; 20(1): 242, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209054

RESUMO

BACKGROUND: Tuberculous pleurisy (TBP) is the most common form of extrapulmonary tuberculosis (TB). However, rapid diagnostic methods with high accuracy for tuberculous pleurisy are urgently needed. In the present study, we evaluated the diagnostic accuracy of Xpert MTB/RIF, LAMP and SAT-TB assay with pleural fluids from culture-positive TBP patients. METHODS: We prospectively enrolled 300 patients with exudative pleural effusions used as the samples for Xpert MTB/RIF, LAMP and SAT-TB assay. Of these, 265 including 223 patients diagnosed with TBP and 42 non-TBP patients used as controls were analyzed. RESULTS: The sensitivities of Xpert MTB/RIF (27.4%), LAMP (26.5%) and SAT-TB assay (32.3%) were significantly higher than that of pleural effusion smear (14.3%, X2 = 20.65, P <  0.001), whereas they were much lower than expected for the analysis of pleural effusion samples. Both SAT-TB assay and Xpert MTB/RIF demonstrated high specificities (100%) and PPVs (100%), but the NPVs of all of the tests were < 22%. The area under ROC curve of pleural effusion smear, LAMP, Xpert MTB/RIF and SAT-TB assays was 0.524 (95% CI 0.431-0.617), 0.632 (95% CI 0.553-0.71), 0.637 (95% CI 0.56-0.714) and 0.673 (95% CI 0.6-0.745). SAT-TB assays had the highest AUC. CONCLUSION: Nucleic acid amplification tests are not the first choice in the diagnosis of tuberculous pleurisy. In this type of test, SAT-TB is recommended because of its low cost, relatively more accurate compared with the other two tests. This prospective study was approved by The Ethics Committee of the Shanghai Pulmonary Hospital (approval number: K19-148). TRIAL REGISTRATION: ClinicalTrials.gov identifier: ChiCTR1900026234 (Retrospectively registered). The registration date is September 28, 2019.


Assuntos
Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Tuberculose Pleural/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , DNA Bacteriano/genética , Confiabilidade dos Dados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/economia , Derrame Pleural/microbiologia , Estudos Prospectivos , RNA Bacteriano/genética , Curva ROC , Sensibilidade e Especificidade , Tuberculose Pleural/microbiologia , Adulto Jovem
17.
Arch Virol ; 165(3): 743-747, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31980939

RESUMO

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/µl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.


Assuntos
Doenças do Gato/virologia , Apresentação Cruzada , Técnicas de Amplificação de Ácido Nucleico/veterinária , Varicellovirus/isolamento & purificação , Proteínas do Envelope Viral/isolamento & purificação , Animais , Doenças do Gato/diagnóstico , Gatos , Primers do DNA/genética , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade
18.
J Agric Food Chem ; 68(3): 899-906, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31891505

RESUMO

Convenient, portable, and low-cost multiplex nucleic acid testing (NAT) systems are the trends in the fields of food safety, environmental microorganisms, molecular diagnosis, etc. In this study, we developed a novel system for visual monitoring of multiple nucleic acids combining a mini-disk capillary array (diameter = 17 mm, embedded with 6-10 capillaries), visual loop-mediated isothermal amplification (LAMP), and quick DNA extraction called mDC-LAMP. The performance and applicability of mDC-LAMP in testing multiple nucleic acids were evaluated and verified employing genetically modified contents analysis as an example. All of the results confirmed that mDC-LAMP has the advantages of high specificity without any cross contamination, high sensitivity with a limit of detection of 25 copies/reaction, high throughput with flexible channel sensors, easy fabrication, and low costs. We believe that mDC-LAMP is a competitive choice for on-spot monitoring of multiple nucleic acids in terms of the easy fabrication/operation, low costs, and suitable performance presented in the nucleic acids test.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , DNA de Plantas/genética , Alimentos Geneticamente Modificados , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/economia , Reação em Cadeia da Polimerase , Zea mays/genética
19.
Am J Trop Med Hyg ; 102(2): 408-414, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31820709

RESUMO

The infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies' portable MinION sequencer. However, the question remains open whether MinION-based bacterial whole genome sequencing is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate of Rickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR, using v9.5 chemistry, and in parallel, we sequenced the same isolate on the Illumina platform in a genomics laboratory in the United Kingdom. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 indel errors. The combined assembly represents the first complete genome sequence of a human R. typhi isolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, with no rearrangements. Filtering based on the known error profile of MinION data improved the accuracy of the nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. Although nanopore-only sequencing cannot yet recover phylogenetic signals in R. typhi, such an approach may be applicable for more diverse organisms.


Assuntos
DNA Bacteriano/genética , Genoma Bacteriano , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Rickettsia typhi/genética , Humanos
20.
PLoS Negl Trop Dis ; 13(11): e0007698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31697673

RESUMO

Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.


Assuntos
Leishmaniose/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Animais , DNA de Protozoário/genética , Bases de Dados Factuais , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Humanos , Leishmania/genética , Programas de Rastreamento/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Psychodidae/parasitologia , Sensibilidade e Especificidade , Fatores de Tempo
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