CODIV-19 et détection moléculaire du SARS-CoV-2 chez les individus asymptomatiques / CODIV-19 and molecular detection of SARS-CoV-2 in asymptomatic individuals

CONTEXTE: Le présent document ainsi que les constats qu'il énonce ont été rédigés en soutien aux travaux du ministère de la Santé et des Services sociaux (MSSS) et de l'Institut national de santé publique du Québec (INSPQ) dans le contexte de l'urgence sanitaire liée à la maladie à coronavirus (COVID-19) au Québec. L'objectif est de réaliser une recension sommaire des données publiées et de mobiliser les savoirs clés afin d'informer les décideurs publics et les professionnels de la santé et des services sociaux. Vu la nature rapide de cette réponse, les constats ou les positions qui en découlent ne reposent pas sur un repérage exhaustif des données publiées, une évaluation de la qualité méthodologique des études avec une méthode systématique ou sur un processus de consultation élaboré. Dans les circonstances d'une telle urgence de santé publique, l'INESSS reste à l'affût de toutes nouvelles données susceptibles de lui faire modifier cette réponse rapide. CONSTATS DE L'INESSS À CE JOUR: Considérant la situation actuelle de pandémie au Québec et ailleurs dans le monde, et à la lumière des informations présentées dans cet état des connaissances sur le diagnostic moléculaire de la COVID-19, l'INESSS dégage les constats suivants: -La charge virale du SARS-CoV-2 évolue dans le temps en fonction du stade de la maladie et influence le résultat du test par RT-PCR. Généralement, les faux négatifs sont attribuables à une charge virale trop faible au moment du prélèvement, surtout si celui-ci a été effectué au tout début ou à la toute fin de l'infection virale. -Un ou plusieurs résultats négatifs n'excluent pas la possibilité d'une infection par le SARS-CoV-2. -Plusieurs enquêtes épidémiologiques suggèrent que des individus auraient été infectés par des personnes pré-symptomatiques, mais aussi par des asymptomatiques. -Des données fragmentaires issues de différentes sources comportant plusieurs limites rapportent que la proportion d'individus asymptomatiques serait importante. Les chiffres disponibles varient en fonction de l'épidémiologie locale, de la population à l'étude et des conditions et critères d'accès au test. -Bien que ne faisant pas consensus, le dépistage systématique de certains groupes de personnes, comme les travailleurs de la santé et les résidents de milieux de soins de longue durée, a été recommandé par quelques organisations et experts afin de contenir la propagation de la maladie et protéger les personnes vulnérables. PRÉSENTATION DE LA DEMANDE: Dans le contexte actuel de pandémie de la COVID-19, des efforts massifs sont investis dans la détection de cette maladie. Dans un premier temps, le présent document traite de la détection moléculaire du virus SARS-CoV-2 par technique d'amplification des acides nucléiques (TAAN), et ce, en ce qui a trait aux situations cliniques à prioriser, aux performances diagnostiques attendues et aux éléments à considérer pour en assurer la fiabilité. Ce document aborde également la situation des cas asymptomatiques, notamment, leur potentiel infectieux, leur proportion et leur contribution à la chaîne de transmission et à la pertinence de les détecter, s'il y a lieu. MÉTHODOLOGIE: 1) Quelles sont les positions et orientations d'organisations savantes et d'autorités de santé en matière de priorisation de l'accès aux tests de détection du SARS-CoV-2? 2) Quelle est la performance de détection du SARS-CoV-2 par RT-PCR relativement au diagnostic de la COVID-19? a) Quels sont les proportions de résultats faussement négatifs? b) Quels sont les éléments susceptibles de nuire à la fiabilité des résultats du test par RT-PCR pour la détection du SARS-CoV-2? 3) Le dépistage systématique des asymptomatiques peut-il être pertinent et si oui, dans quelles situations? a) Quel est le potentiel infectieux des asymptomatiques (et pré-symptomatiques) et ceux présentant des formes légères de la maladie? b) Quelles sont les proportions d'asymptomatiques et de cas subcliniques dans la communauté? c) Dans quels milieux ou dans quelles situations serait-il bénéfique d'effectuer le dépistage systématique de la COVID-19? SOMMAIRE DES RÉSULTATS: 1. Positions et orientations de sociétés savantes et d'autorités de santé concernant l'utilisation du test moléculaire pour la détection du SARS-CoV-2. En date du 22 avril, vingt-et-un (21) documents en lien avec la pertinence et les critères de détection moléculaire de la COVID-19 adoptés par les autorités de santé d'autres juridictions ont été recensés. Les principaux renseignements extraits de ces documents sont les suivants: -Personnes devant systématiquement être testées ou testées en priorité; -Particularités pour les travailleurs de la santé; -Limites du test à considérer. Ces éléments sont résumés dans les paragraphes suivants. 2. Fiabilité des tests moléculaires pour la détection du SARS-CoV-2 et le diagnostic de la COVID-19 Onze (11) publications traitant de la fiabilité de la détection moléculaire du SARS-CoV-2 ont été retenues, dont : -cinq (5) présentant des proportions de faux négatifs dans le temps [Li Y.; Li D.; Long; Xie; Ai, 2020] et; -six (6) abordant certains éléments susceptibles de nuire à la qualité des résultats de la RT-PCR [Pan; Loeffelholz & Tang; Lippi; Wölfel; Lo; Wikramaratna, 2020]. 3. Potentiel infectieux des individus asymptomatiques et pré-symptomatiques Quinze (15) publications traitant du potentiel infectieux des asymptomatiques, pré-symptomatiques et de formes légères de la maladie ont été retenues.

Solicitud de incorporación de nuevo equipamiento para diagnóstico microbiológico de la tuberculosis / Application for the incorporation of new equipment for the microbiological diagnosis of tuberculosis

INTRODUCIÓN: La tuberculosis (en adelante TB) es una enfermedad causada por Mycobacterium tuberculosis, una bacteria transmitida principalmente por la inhalación de microgotas expelidas al toser, hablar y respirar. Según su localización anatómica puede clasificarse en TB pulmonar y extrapulmonar, también se divide según la co-infección con VIH y la resistencia a medicamentos. La TB es una de las principales causas de mortalidad en el mundo. En 2016, 10,4 millones de personas enfermaron de TB y 1,7 millones murieron por esta enfermedad (entre ellas 0,4 millones de personas con VIH). Más del 95% de las muertes ocurre en países de ingresos bajos y medianos. En Argentina se considera que la enfermedad tiene una carga moderada. En 2016 se notificaron 11560 casos con aumento de la tasa de notificación de 24,9 a 26,5 por 100000 habitantes. Esto determinó el aumento sostenido de la enfermedad, a lo que se sumó un incremento de casos de TB en grupos jóvenes. La provincia de Neuquén es una población de baja endemia con 50 casos incidentes anuales y con un 5 % de casos nuevos o recaídas. Casi no hay reportes de casos de multi-resistencia a drogas. El rango etario más afectado es el de 45 a 64 años, quienes podrían diseminar la enfermedad en caso de ser bacilíferos. EVALUACIÓN DE TECNOLOGÍAS: A partir de un pedido de incorporación se analizaron dos tecnologías principales y se revisaron las recomendaciones y factibilidad de incorporar otras tecnologías en distintos niveles de complejidad para mejorar la detección precoz de Mycobacterium tuberculosis. Se trabajó en la priorización de tecnologías, en la distribución actual con enfoque de mapa sanitario y analizando la organización del sistema de salud y la red prestacional de laboratorio. Las principales tecnologías sanitarias solicitadas fueron: Cultivo en medio líquido, BACTEC MGIT de BECKTON DICKINSON: tecnología fluorescente basada en el consumo de oxígeno para la detección del crecimiento bacteriano del complejo M. tuberculosis. Xpert MTB/RIF de Cepheid: tecnología de amplificación de ácidos nucleicos para la detección del complejo M. tuberculosis y la resistencia a la rifampicina. METODOLOGÍA: Se conformó un equipo multidisciplinario e independiente que luego de diversas reuniones definió preguntas de investigación que abarcó el informe. BÚSQUEDA Y ANÁLISIS DE LA EVIDENCIA CIENTÍFICA: Se realizó una búsqueda no sistemática de bibliografía científica priorizando la inclusión de revisiones sistemáticas y meta-análisis, evaluaciones de tecnologías sanitarias e informes de seguridad, guías OMS y otras Guías de Práctica Clínica basadas en la evidencia. CONSENSO: Se mantuvieron reuniones y rondas de preguntas por mail para lograr consenso sobre los aspectos donde variaban las opiniones. CONCLUSIONES: 1. Se recomienda mejorar la calidad del sistema de información clínica, de laboratorio y epidemiológica. En el 2019 se planifica la mejora del sistema informático junto com proyectos de capacitaciones a personal de salud de toda la provincia para impulsar la notificación de todos los casos. 2. Se recomienda implementar microscopía de fluorescencia en los laboratorios de los hospitales cabeceras de zona según factibilidad. 3. Realizar capacitaciones al personal de salud de las diversas zonas para estimular el pedido de baciloscopías, el adecuado seguimiento de contactos y de tratamientos. 4. A pesar de que la tecnología Gene Xpert muestre numerosas ventajas con respecto al BACTEC MGIT, se trata de un método muy costoso; Teniendo en cuenta que nuestra provincia no es una zona endémica ni presenta resistencia a fármacos se recomienda a favor de la implementación del cultivo en medio líquido automatizado, BACTEC MGIT de BECKTON DICKINSON.

SD-chip enabled quantitative detection of HIV RNA using digital nucleic acid sequence-based amplification (dNASBA).

Quantitative detection of RNA is important in molecular biology and clinical diagnostics. Nucleic acid sequence-based amplification (NASBA), a single-step method to amplify single-stranded RNA, is attractive for use in point-of-care (POC) diagnostics because it is an isothermal technique that is as sensitive as RT-PCR with a shorter reaction time. However, NASBA is limited in its ability to provide accurate quantitative information, such as viral load or RNA copy number. Here we test a digital format of NASBA (dNASBA) using a self-digitization (SD) chip platform, and apply it to quantifying HIV-1 RNA. We demonstrate that dNASBA is more sensitive and accurate than the real-time quantitative NASBA, and can be used to quantify HIV-1 RNA in plasma samples. Digital NASBA is thus a promising POC diagnostics tool for use in resource-limited settings.

A novel portable filtration system for sampling and concentration of microorganisms: Demonstration on marine microalgae with subsequent quantification using IC-NASBA.

This paper presents a novel portable sample filtration/concentration system, designed for use on samples of microorganisms with very low cell concentrations and large volumes, such as water-borne parasites, pathogens associated with faecal matter, or toxic phytoplankton. The example application used for demonstration was the in-field collection and concentration of microalgae from seawater samples. This type of organism is responsible for Harmful Algal Blooms (HABs), an example of which is commonly referred to as "red tides", which are typically the result of rapid proliferation and high biomass accumulation of harmful microalgal species in the water column or at the sea surface. For instance, Karenia brevis red tides are the cause of aquatic organism mortality and persistent blooms may cause widespread die-offs of populations of other organisms including vertebrates. In order to respond to, and adequately manage HABs, monitoring of toxic microalgae is required and large-volume sample concentrators would be a useful tool for in situ monitoring of HABs. The filtering system presented in this work enables consistent sample collection and concentration from 1 L to 1 mL in five minutes, allowing for subsequent benchtop sample extraction and analysis using molecular methods such as NASBA and IC-NASBA. The microalga Tetraselmis suecica was successfully detected at concentrations ranging from 2 × 105 cells/L to 20 cells/L. Karenia brevis was also detected and quantified at concentrations between 10 cells/L and 106 cells/L. Further analysis showed that the filter system, which concentrates cells from very large volumes with consequently more reliable sampling, produced samples that were more consistent than the independent non-filtered samples (benchtop controls), with a logarithmic dependency on increasing cell numbers. This filtering system provides simple, rapid, and consistent sample collection and concentration for further analysis, and could be applied to a wide range of different samples and target organisms in situations lacking laboratories.

Universal nucleic acid sequence-based amplification for simultaneous amplification of messengerRNAs and microRNAs.

A universal NASBA assay is presented for simultaneous amplification of multiple microRNA (miRNA) and messengerRNA (mRNA) sequences. First, miRNA and mRNA sequences are reverse transcribed using tailed reverse transcription primer pairs containing a gene-specific and an non-specific region. For reverse transcription of small miRNA molecules a non-specific region is incorporated into a structured stem-loop reverse transcription primer. Second, a universal NASBA primer pair that recognizes the tagged cDNA molecules enables a simultaneous, transcription-based amplification reaction (NASBA) of all different cDNA molecules in one reaction. The NASBA products (RNA copies) are detected by gene-specific DNA probes immobilized on a biochip. By using the multiplex reverse transcription combined with the universal NASBA amplification up to 14 different mRNA and miRNA sequences can be specifically amplified and detected in parallel. In comparison with standard multiplex NASBA assays this approach strongly enhances the multiplex capacity of NASBA-based amplification reactions. Furthermore simultaneous assaying of different RNA classes can be achieved that might be beneficial for studying miRNA-based regulation of gene expression or for RNA-based tumor diagnostics.

Multifunctional sample preparation kit and on-chip quantitative nucleic acid sequence-based amplification tests for microbial detection.

This study reports a quantitative nucleic acid sequence-based amplification (Q-NASBA) microfluidic platform composed of a membrane-based sampling module, a sample preparation cassette, and a 24-channel Q-NASBA chip for environmental investigations on aquatic microorganisms. This low-cost and highly efficient sampling module, having seamless connection with the subsequent steps of sample preparation and quantitative detection, is designed for the collection of microbial communities from aquatic environments. Eight kinds of commercial membrane filters are relevantly analyzed using Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus aureus as model microorganisms. After the microorganisms are concentrated on the membrane filters, the retentate can be easily conserved in a transport medium (TM) buffer and sent to a remote laboratory. A Q-NASBA-oriented sample preparation cassette is originally designed to extract DNA/RNA molecules directly from the captured cells on the membranes. Sequentially, the extract is analyzed within Q-NASBA chips that are compatible with common microplate readers in laboratories. Particularly, a novel analytical algorithmic method is developed for simple but robust on-chip Q-NASBA assays. The reported multifunctional microfluidic system could detect a few microorganisms quantitatively and simultaneously. Further research should be conducted to simplify and standardize ecological investigations on aquatic environments.

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis.

Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (∼100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.

Microfluidic reactors for diagnostics applications.

Diagnostic assays are an important part of health care, both in the clinic and in research laboratories. In addition to improving treatments and clinical outcomes, rapid and reliable diagnostics help track disease epidemiology, curb infectious outbreaks, and further the understanding of chronic illness. Disease markers such as antigens, RNA, and DNA are present at low concentrations in biological samples, such that the majority of diagnostic assays rely on an amplification reaction before detection is possible. Ideally, these amplification reactions would be sensitive, specific, inexpensive, rapid, integrated, and automated. Microfluidic technology currently in development offers many advantages over conventional benchtop reactions that help achieve these goals. The small reaction volumes and energy consumption make reactions cheaper and more efficient in a microfluidic reactor. Additionally, the channel architecture could be designed to perform multiple tests or experimental steps on one integrated, automated platform. This review explores the current research on microfluidic reactors designed to aid diagnostic applications, covering a broad spectrum of amplification techniques and designs.

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process.

Use of an HIV-1 reverse-transcriptase enzyme-activity assay to measure HIV-1 viral load as a potential alternative to nucleic acid-based assay for monitoring antiretroviral therapy in resource-limited settings.

An inexpensive and technically less-demanding methodology to quantify HIV-1 viral load would be of great value for resource-limited settings, where the nucleic-acid amplification technique (NAAT) is impractical and/or resource-prohibitive. In this study, an HIV-1 reverse-transcriptase enzyme-activity assay (ExaVir Load assay, version 1) was compared with the gold standard RT-PCR assay, Roche HIV-1 Amplicor Monitor, version 1.5. A total of 121 plasma specimens were used for the evaluation. ExaVir Load had a sensitivity of 97 % and a specificity of 71 % in identifying specimens with <400 copies ml(-1) in the Roche RT-PCR assay as being less than the detection limit of the assay (5500 copies ml(-1)). The mean difference (95 % limits of agreement) between Roche RT-PCR and ExaVir Load was -0.23 (-1.59 to 1.13) log(10)(copies ml(-1)) by Bland-Altman analysis. Significant negative correlations were seen between CD4(+) T-cell counts and the ExaVir Load assay (r=-0.32, P<0.05), and between CD4(+) T-cell counts and the Roche RT-PCR (r=-0.38, P<0.01). The present study with HIV-1 showed a strong correlation between the ExaVir Load assay and the RT-PCR assay. Hence, the ExaVir Load assay could be considered for use in resource-limited settings as an alternative viral-load assay to the standard NAAT-based assay after further evaluation with prospective specimens.

Practical experiences of moving molecular diagnostics into routine use at the Veterinary Laboratories Agency.

The practical bench application of molecular tests (here defined as nucleic acid-based tests) in a routine testing situation is not without its challenges. This paper outlines the approaches we take at the Veterinary Laboratories Agency (VLA) and highlights some of the practical issues which we have found to be important for the successful introduction and use of molecular tests in a routine testing environment. The potential advantages of molecular tests, and factors which dictate which tests are adopted for routine testing, are discussed before giving some examples of molecular tests in routine use at the VLA. The instrumentation, reagents and assays we use are outlined, followed by sections of how we approach validation and how we manage and resource the transfer of molecular tests into routine use.

Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1.

We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification (NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant recombinant viruses and all group M HIV-1 subtypes.

Characteristics and applications of nucleic acid sequence-based amplification (NASBA).

Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only in the absence of the corresponding RNA target or in case of an excess (>1,000-fold) of target DNA over RNA. As NASBA is primer-dependent and amplicon detection is based on probe binding, primer and probe design rules are included. An overview of various target nucleic acids that have been amplified successfully using NASBA is presented. For the isolation of nucleic acids prior to NASBA, the "Boom" method, based on the denaturing properties of guanidine isothiocyanate and binding of nucleic acid to silica particles, is preferred. Currently, electro-chemiluminescence (ECL) is recommended for the detection of the amplicon at the end of amplification. In the near future, molecular beacons will be introduced enabling "real-time detection," i.e., amplicon detection during amplification. Quantitative HIV-1 NASBA and detection of up to 48 samples can then be performed in only 90 min.