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1.
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
2.
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
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.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
Value Health ; 23(2): 180-190, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32113623

RESUMO

OBJECTIVES: Direct-acting antivirals containing nonstructural protein 5A (NS5A) inhibitors administered over 8 to 12 weeks are effective in ∼95% of patients with hepatitis C virus. Nevertheless, patients resistant to NS5A inhibitors have lower cure rates over 8 weeks (<85%); for these patients, 12 weeks of treatment produces cure rates greater than 95%. We evaluated the lifetime cost-effectiveness of testing for NS5A resistance at baseline and optimizing treatment duration accordingly in genotype 1 noncirrhotic treatment-naïve patients from the perspective of the UK National Health Service. METHODS: A decision-analytic model compared (1) standard 12-week treatment (no testing), (2) shortened 8-week treatment (no testing), and (3) baseline testing with 12-/8-week treatment for those with/without NS5A polymorphisms. Patients who failed first-line therapy were retreated for 12 weeks. Model inputs were derived from published studies. Costs, quality-adjusted life-years, and the probability of cost-effectiveness were calculated. RESULTS: Baseline testing had an incremental net monetary benefit (INMB) of £11 838 versus standard 12 weeks of therapy (no testing) and low probability (31%) of being the most cost-effective, assuming £30 000 willingness to pay. Shortened 8 weeks of treatment (no testing) had an INMB of £12 294 and the highest probability (69%) of being most cost-effective. Scenario analyses showed baseline testing generally had the highest INMB and probability of being most cost-effective if first- and second-line drug prices were low (<£20k). CONCLUSIONS: Optimizing treatment duration based on NS5A polymorphisms for genotype 1 noncirrhotic treatment-naive patients in the United Kingdom is not cost-effective if the drug costs are high; the strategy is generally most cost-effective when drug prices are low (<£20k).


Assuntos
Antivirais/economia , Antivirais/uso terapêutico , Custos de Medicamentos , Farmacorresistência Viral , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/economia , Técnicas de Diagnóstico Molecular/economia , Polimorfismo Genético , Proteínas não Estruturais Virais/antagonistas & inibidores , Adulto , Antivirais/efeitos adversos , Análise Custo-Benefício , Técnicas de Apoio para a Decisão , Árvores de Decisões , Farmacorresistência Viral/genética , Feminino , Genótipo , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/virologia , Humanos , Masculino , Cadeias de Markov , Modelos Econômicos , Terapia de Alvo Molecular/economia , Valor Preditivo dos Testes , Anos de Vida Ajustados por Qualidade de Vida , Medicina Estatal/economia , Fatores de Tempo , Resultado do Tratamento , Reino Unido , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
12.
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
14.
BMC Infect Dis ; 20(1): 170, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087681

RESUMO

BACKGROUND: Infectious meningitis is a serious disease and patient outcome relies on fast and reliable diagnostics. A syndromic panel testing approach like the FilmArray ME can accelerate diagnosis and therefore decrease the time to pathogen specific therapy. Yet, its clinical utility is controversial, mainly because of a remaining uncertainty in correct interpretation of results, limited data on its performance on clinical specimens and its relatively high costs. The aim of this study was to analyze clinical performance of the assay in a real life setting at a tertiary university hospital using a pragmatic and simple sample selection strategy to reduce the overall cost burden. METHODS: Over a period of 18 months we received 4623 CSF samples (2338 hospitalizations, 1601 individuals). FilmArray ME analysis was restricted to CSF-samples with a high pretest probability of infectious meningitis, e.g. positive Gram-stain, samples in which leukocytes and/or bacteria were evident or urgent suspicion of infection was communicated by clinicians. N = 171 samples matched to our risk criteria and were subjected to FilmArray ME analysis. Those samples were also analyzed by reference methods: culture only (n = 45), PCR only (n = 20) or both methods (n = 106). RESULTS: 56/171 (32.75%) were FilmArray ME positive. Bacterial pathogens were detected in 30/56 (53.57%), viral pathogens were detected in 27/56 (48.21%) and yeast DNA was detected in 1/56 (1.79%) of positive samples. Double detection occurred in 2/56 samples. In 52/56 (92.86%) FilmArray ME positive samples, results could be confirmed by the reference assays (sensitivity = 96.30%, specificity =96.58%). CONCLUSION: The FilmArray ME assay is a fast and reliable diagnostic tool for the management of infectious meningitis and can easily be implemented in routine diagnostic workflows. However, correlation of test results and underlying clinical symptoms requires experienced users and the awareness of potentially false negative or false positive results. Moreover, considering the need for antimicrobial susceptibility testing, the use of molecular tests as a stand-alone diagnostic cannot be recommended.


Assuntos
Testes Diagnósticos de Rotina/métodos , Encefalite/diagnóstico , Meningite/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Coloração e Rotulagem/métodos , Testes Diagnósticos de Rotina/economia , Encefalite/líquido cefalorraquidiano , Encefalite/microbiologia , Encefalite/virologia , Violeta Genciana , Alemanha , Hospitais Universitários , Humanos , Laboratórios , Meningite/líquido cefalorraquidiano , Meningite/microbiologia , Meningite/virologia , Técnicas de Diagnóstico Molecular/economia , Reação em Cadeia da Polimerase Multiplex/economia , Fenazinas , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/economia , Centros de Atenção Terciária
15.
BMC Infect Dis ; 20(1): 38, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937247

RESUMO

BACKGROUND: Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC therefore recommends GBS screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. METHOD: In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, Revogene GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsin's clinical microbiology laboratory for GBS testing. RESULTS: We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with a positive percent agreement of 98.0% and a negative percent agreement of 96.5% between the Revogene GBS LB assay and the GeneXpert GBS LB assay. CONCLUSION: Overall, we report that both assays perform well for the detection of GBS colonization in pregnant women.


Assuntos
Testes Diagnósticos de Rotina/métodos , Programas de Rastreamento/métodos , Técnicas de Diagnóstico Molecular/métodos , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/métodos , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/genética , DNA Viral/análise , Feminino , Técnicas Genéticas , Humanos , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Programas de Rastreamento/economia , Técnicas de Diagnóstico Molecular/economia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Gestantes , Infecções Estreptocócicas/virologia , Fatores de Tempo , Vagina/virologia
16.
PLoS One ; 15(1): e0227138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999746

RESUMO

BACKGROUND: Tuberculosis (TB) is the leading cause of death among HIV-positive patients. We assessed the cost-effectiveness of including lateral-flow urine lipoarabinomannan (LF-LAM) in TB diagnostic algorithms for severely ill or immunosuppressed HIV-positive patients with symptoms of TB in Kenya. METHODS: From a decision-analysis tree, ten diagnostic algorithms were elaborated and compared. All algorithms included clinical exam. The costs of each algorithm were calculated using a 'micro-costing' method. The efficacy was estimated through a prospective study that included severely ill or immunosuppressed (CD4<200cells/µL) HIV-positive adults with symptoms of TB. The cost-effectiveness analysis was performed using the disability-adjusted life year (DALY) averted as effectiveness outcome. A 4% discount rate was applied. RESULTS: The algorithm that added LF-LAM alone to the clinical exam lead to the least average cost per TB case detected (€47) and was the most cost-effective with a cost/DALY averted of €4.6. The algorithms including LF-LAM, microscopy and X-ray, and LF-LAM and Xpert in sputum, detected a high number of TB cases with a cost/DALY averted of €6.1 for each of them. In the comparisons of the algorithms two by two, using LF-LAM instead of microscopy (clinic&LAM vs clinicµscopy) and using LF-LAM along with GeneXpert in sputum instead of GeneXpert in urine along with GeneXpert in sputum, (clinic&LAM&Xpert_sputum vs clinic&Xpert_sputum&Xpert_urine) led to the highest increase in the cost-effectiveness ratios (ICERs): €-7.2 and €-12.6 respectively. In these two comparisons, using LF-LAM increased the number of TB patients detected while reducing costs. Adding LF-LAM to smear microscopy alone or to smear microscopy and Xray led to the highest increase in the additional number of TB cases detected (31 and 25 respectively) with an incremental efficiency estimated at 134 and 344 DALYs respectively. The ICERs were €22.0 and €8.6 respectively. CONCLUSION: Including LF-LAM in TB diagnostic algorithms is cost-effective for severely ill or immunosuppressed HIV-positive patients.


Assuntos
Análise Custo-Benefício , Infecções por HIV/complicações , Lipopolissacarídeos/urina , Técnicas de Diagnóstico Molecular/economia , Tuberculose/diagnóstico , Adulto , Biomarcadores/urina , Feminino , Humanos , Masculino , Tuberculose/urina
18.
Parasit Vectors ; 12(1): 443, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31522691

RESUMO

BACKGROUND: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. RESULTS: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a 'pooling approach' can yield a low frequency of 'missed' infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. CONCLUSIONS: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in 'pools-of-five'. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.


Assuntos
Testes Diagnósticos de Rotina/métodos , Fezes/parasitologia , Helmintíase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Manejo de Espécimes/métodos , Custos e Análise de Custo , Testes Diagnósticos de Rotina/economia , Humanos , Técnicas de Diagnóstico Molecular/economia , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Manejo de Espécimes/economia
20.
Genes (Basel) ; 10(9)2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470607

RESUMO

Enteroviruses affect millions of people worldwide and are of significant clinical importance. The standard method for enterovirus identification and genotyping still relies on Sanger sequencing of short diagnostic amplicons. In this study, we assessed the feasibility of nanopore sequencing using the new flow cell "Flongle" for fast, cost-effective, and accurate genotyping of human enteroviruses from clinical samples. PCR amplification of partial VP1 gene was performed from multiple patient samples, which were multiplexed together after barcoding PCR and sequenced multiple times on Flongle flow cells. The nanopore consensus sequences obtained from mapping reads to a reference database were compared to their Sanger sequence counterparts. Using clinical specimens sampled over different years, we were able to correctly identify enterovirus species and genotypes for all tested samples, even when doubling the number of barcoded samples on one flow cell. Average sequence identity across sequencing runs was >99.7%. Phylogenetic analysis showed that the consensus sequences achieved with Flongle delivered accurate genotyping. We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping.


Assuntos
Enterovirus/genética , Técnicas de Genotipagem/métodos , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de RNA/métodos , Sequência Consenso , Técnicas de Genotipagem/economia , Técnicas de Genotipagem/instrumentação , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Nanoporos , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/instrumentação , Proteínas Virais de Fusão/genética
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