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1.
Curr Oncol ; 28(1): 847-852, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33567626

RESUMEN

The pandemic of SARS-CoV-2 is a serious global challenge affecting millions of people worldwide. Cancer patients are at risk for infection exposure and serious complications. A prompt diagnosis of SARS-CoV-2 infection is crucial for the timely adoption of isolation measures and the appropriate management of cancer treatments. In lung cancer patients the symptoms of infection 19 may resemble those exhibited by the underlying oncologic condition, possibly leading to diagnostic overlap and delays. Moreover, cancer patients might display a prolonged positivity of nasopharyngeal RT-PCR assays for SARS-CoV-2, causing long interruptions or delay of cancer treatments. However, the association between the positivity of RT-PCR assays and the patient's infectivity remains uncertain. We describe the case of a patient with non-small cell lung cancer, and a severe ab extrinseco compression of the trachea, whose palliative radiotherapy was delayed because of the prolonged positivity of nasopharyngeal swabs for SARS-CoV-2. The patient did not show clinical symptoms suggestive of active infection, but the persistent positivity of RT-PCR assays imposed the continuation of isolation measures and the delay of radiotherapy for over two months. Finally, the negative result of SARS-CoV-2 viral culture allowed us to verify the absence of viral activity and to rule out the infectivity of the patient, who could finally continue her cancer treatment.


Asunto(s)
/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/virología , Neoplasias Pulmonares/virología , /aislamiento & purificación , Anciano , Portador Sano/diagnóstico , Femenino , Humanos , Nasofaringe/virología , ARN Viral/genética , Tiempo de Tratamiento
2.
Nat Commun ; 12(1): 776, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536425

RESUMEN

The rapid expansion of the COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health and economic priority. Taking advantage of versatility and rapid development, three SARS-CoV-2 mRNA vaccine candidates have entered clinical trials with a two-dose immunization regimen. However, the waning antibody response in convalescent patients after SARS-CoV-2 infection and the emergence of human re-infection have raised widespread concerns about a possible short duration of SARS-CoV-2 vaccine protection. Here, we developed a nucleoside-modified mRNA vaccine in lipid-encapsulated form that encoded the SARS-CoV-2 RBD, termed as mRNA-RBD. A single immunization of mRNA-RBD elicited both robust neutralizing antibody and cellular responses, and conferred a near-complete protection against wild SARS-CoV-2 infection in the lungs of hACE2 transgenic mice. Noticeably, the high levels of neutralizing antibodies in BALB/c mice induced by mRNA-RBD vaccination were maintained for at least 6.5 months and conferred a long-term notable protection for hACE2 transgenic mice against SARS-CoV-2 infection in a sera transfer study. These data demonstrated that a single dose of mRNA-RBD provided long-term protection against SARS-CoV-2 challenge.


Asunto(s)
/inmunología , /inmunología , /genética , /inmunología , /genética , Animales , Anticuerpos Neutralizantes/inmunología , /genética , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Pandemias/prevención & control , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Viral/genética , ARN Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunología
3.
Genome Med ; 13(1): 21, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563320

RESUMEN

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.


Asunto(s)
/genética , Genoma Viral/genética , Pandemias , /genética , /virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Viral/genética , Secuenciación Completa del Genoma
4.
PLoS One ; 16(2): e0246867, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33566873

RESUMEN

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.


Asunto(s)
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , ARN Viral/genética , /aislamiento & purificación , Humanos , Límite de Detección , Tamizaje Masivo , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Saliva/virología , Manejo de Especímenes , Factores de Tiempo
5.
Genet Test Mol Biomarkers ; 25(2): 85-101, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33596144

RESUMEN

Coronavirus disease 2019 (COVID-19) displays a broad spectrum of clinical presentations ranging from lack of symptoms to severe multiorgan system complications and death. Various laboratory assays have been employed in the diagnosis of COVID-19, including: nucleic acid-based tests; antigen tests; and serum testing for anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies. The disease can also be diagnosed based on suggestive clinical features and radiological findings. Until now, remdesivir is the only medication approved for the treatment of COVID-19 by the U.S. Food and Drug Administration (FDA); however, it is anticipated that several anti-SARS-CoV-2 monoclonal antibodies will gain soon approval. Other methods of treatment include supportive care directed toward treating the symptoms. Nevertheless, many studies have recently emerged, showing controversial preliminary results with the off-label medication hydroxychloroquine. Given that all results are still preliminary, including those seen by remdesivir, additional evidence and research are required to identify effective medications that are broadly effective and well tolerated. Importantly, two RNA-based vaccines have recently gained approval from Pfizer and Moderna, with many others still in clinical trials. This article reviews various aspects of COVID-19, including its epidemiology; its evolution and mutational spectrum; and its clinical dynamics, symptoms and complications, diagnosis, and treatment.


Asunto(s)
Carga Global de Enfermedades/estadística & datos numéricos , Pandemias/estadística & datos numéricos , /patogenicidad , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/uso terapéutico , Antivirales/uso terapéutico , /tratamiento farmacológico , /terapia , /métodos , Ensayos Clínicos como Asunto , Evolución Molecular , Humanos , Hidroxicloroquina/uso terapéutico , Mutación , Uso Fuera de lo Indicado , Pandemias/prevención & control , ARN Viral/genética , ARN Viral/aislamiento & purificación , /inmunología , Índice de Severidad de la Enfermedad
6.
Viruses ; 13(2)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33530363

RESUMEN

The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.


Asunto(s)
VIH-1/genética , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Empalme Alternativo , Exones , Regulación Viral de la Expresión Génica , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genética , Secuencias Reguladoras de Ácidos Nucleicos , Latencia del Virus/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo
7.
Viruses ; 13(2)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33530573

RESUMEN

Sapovirus is a common cause of acute gastroenteritis in all age groups. Sapovirus infections are seldom investigated in Spain, and its epidemiology in the country is not well known. The use of molecular diagnostic procedures has allowed a more frequent detection of sapoviruses in patients with diarrhea. A total of 2545 stool samples from patients with acute gastroenteritis attended from June 2018 to February 2020 at the Clinic University Hospital in Valencia, Spain, were analyzed by reverse transcription (RT) and real-time multiplex PCR (RT-PCR) to investigate the etiology of enteric infections. Sapovirus was the second enteric virus detected with a positive rate of 8%, behind norovirus (12.2%) and ahead of rotavirus (7.1%), astrovirus (4.9%) and enteric adenoviruses (2.9%). Most sapovirus infections occurred in infants and young children under 3 years of age (74%) with the highest prevalence in autumn and early winter. Coinfections were found in 25% of the patients with sapovirus diarrhea, mainly with other enteric viruses. Genotyping demonstrated the circulation of seven different genotypes during the study period, with a predominance of genotypes GI.1, GI.2, and GII.1. Phylogenetic analysis showed that genogroup GII strains form a cluster separated from genogroup GI and GV, being genotype GV.1 strains related to genotype GI.1 and GI.2 strains.


Asunto(s)
Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Gastroenteritis/epidemiología , Gastroenteritis/virología , Sapovirus/genética , Factores de Edad , Infecciones por Caliciviridae/diagnóstico , Coinfección/epidemiología , Coinfección/microbiología , Coinfección/virología , Diarrea/diagnóstico , Diarrea/epidemiología , Diarrea/virología , Femenino , Gastroenteritis/diagnóstico , Variación Genética , Genotipo , Humanos , Masculino , Epidemiología Molecular , Reacción en Cadena de la Polimerasa Multiplex , Filogenia , Prevalencia , ARN Viral/genética , Sapovirus/clasificación , Sapovirus/aislamiento & purificación , Estaciones del Año , España/epidemiología
8.
Viruses ; 13(2)2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33546342

RESUMEN

Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR; confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.


Asunto(s)
Animales Salvajes/virología , Artiodáctilos/virología , Orthoreovirus de los Mamíferos/aislamiento & purificación , Animales , Animales Salvajes/clasificación , Artiodáctilos/clasificación , Reservorios de Enfermedades/veterinaria , Reservorios de Enfermedades/virología , Italia/epidemiología , Hígado/virología , Orthoreovirus de los Mamíferos/genética , Prevalencia , ARN Viral/genética , Serogrupo
9.
Mol Aspects Med ; 77: 100943, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33551236

RESUMEN

The health of the individual and the population in general is the result of interaction between genetics and various environmental factors, of which diet/nutrition is the most important. The focus of this paper is on the association of high n-6 PUFA or low n-3 PUFA due to genetic variation and/or dietary intake, with changes in specialized pro-resolving mediators (SPMs), cytokine storm, inflammation-resolution and Covid-19. Human beings evolved on a diet that was balanced in the n-6 and n-3 essential fatty acids with a ratio of n-6/n-3 of 1-2/1 whereas today this ratio is 16/1. Such a high ratio due to high amounts of n-6 fatty acids leads to a prothrombotic and proinflammatory state and is associated with obesity, diabetes, cardiovascular disease, and some forms of cancer. In addition to the high intake of n-6 fatty acids that increases inflammation there is genetic variation in the biosynthesis of n-6 linoleic acid (LA) to arachidonic acid (ARA) and of linolenic (ALA) to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Present day humans have two common FADS haplotypes that differ dramatically in their ability to generate long-chain fatty acids. The more efficient, evolutionary derived haplotype increases the efficiency of synthesizing essential long-chain fatty acids from precursors and could have provided an advantage in environments with limited access to dietary long-chain fatty acids ARA, EPA and DHA. In the modern world this haplotype has been associated with lifestyle-related diseases, such as cardiovascular disease, obesity, diabetes, all of which are characterized by increased levels of inflammation. African Americans and Latino populations have increased susceptibility and higher death rates from SARS-CoV-2 than whites. These populations are characterized by increased numbers of persons (about 80%) that are fast metabolizers, leading to increased production of ARA, as well as poor intake of fruits and vegetables. The combinations of fast metabolism and high n-6 intake increases their inflammatory status and possibly susceptibility of SARS-CoV-2. In vitro and human studies indicate that the specialized pro-resolving mediators (SPM) produced from the n-3, EPA and DHA influence the resolution of inflammation, allowing the tissues to return to function and homeostasis. The SPMs each counter-regulate cytokine storms, as well as proinflammatory lipid mediators via NFκB and inflammasome down regulation and reduce the proinflammatory eicosanoids produced from ARA. The nutritional availability of dietary n-3 fatty acids from marine oils enriched with SPM intermediate precursors, along with increasing local biosynthesis of SPMs to functional concentrations may be an approach of value during SARS-CoV2 infections, as well as in prevention, and shortening their recovery from infections. It is evident that populations differ in their genetic variants and their frequencies and their interactions with the food they eat. Gene-nutrient interactions is a very important area of study that provides specific dietary advice for individuals and subgroups within a population in the form of Precision Nutrition. Nutritional science needs to focus on Precision Nutrition, genetic variants in the population and a food supply composed of Nutrients that have been part of our diet throughout evolution, which is the diet that our genes are programmed to respond.


Asunto(s)
/dietoterapia , /genética , /metabolismo , Ácidos Docosahexaenoicos/metabolismo , Eicosanoides/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Ácidos Grasos Esenciales/metabolismo , Ácidos Grasos Omega-3/metabolismo , Predisposición Genética a la Enfermedad/genética , Haplotipos , Humanos , Inflamación/dietoterapia , Inflamación/genética , Inflamación/metabolismo , Ácido Linoleico/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , /patogenicidad
10.
Viruses ; 13(2)2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33562806

RESUMEN

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017-2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.


Asunto(s)
Enterovirus/genética , Enterovirus/aislamiento & purificación , Microbiología del Agua , Proteínas de la Cápside/genética , Enterovirus/clasificación , Monitoreo del Ambiente , Genoma Viral/genética , Humanos , Nigeria , Filogenia , ARN Viral/genética , Recombinación Genética , Serogrupo , Aguas del Alcantarillado/virología
11.
Viruses ; 13(2)2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33573092

RESUMEN

Phleboviruses transmitted by phlebotomine sandflies are endemic in the Mediterranean basin. Toscana phlebovirus (TOSV), Sicilian phlebovirus (SFSV), and Naples phlebovirus (SFNV) are responsible of summer fever, with well-known pathogenic potential for humans ranging from asymptomatic to mild fever, in addition to neuro-invasive infections during summer. Although TOSV, in particular, is a significant and well-known human pathogen, SFVs remain neglected, with many gaps in the relevant knowledge. Sero-epidemiological studies and case reports recently showed a geographical wider distribution than previously considered, although the real incidence of phleboviruses infections in the Mediterranean area is still unknown. Here we retrospectively evaluated the circulation of phleboviruses during summer seasons between 2007 and 2019 in 649 patients showing neurological symptoms using both molecular and serological approaches. We found that 42/649 (6.5%) subjects experienced phlebovirus infection and only 10/42 cases were detected by molecular assays, whereas the other 32/42 were identified using serological approaches, including neutralization assays. During the 2013 summer, an outbreak in the Lombardy region is described because the prevalence of phlebovirus infection reached 37.2% (19/51 subjects). Interestingly, only 5/19 (26.5%) reported traveling in endemic areas. Of note, no cross-neutralization was observed between different strains tested, showing the possibility to be reinfected by newly discovered phlebovirus strains. In conclusion, phlebovirus infections are still inadequately considered by physicians and are generally underestimated. However, based on our results, sandfly fever viruses should be routinely included in diagnostic panels during summer period, including in Northern Italy.


Asunto(s)
Fiebre por Flebótomos/diagnóstico , Fiebre por Flebótomos/epidemiología , Phlebovirus/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Neutralizantes/sangre , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Italia/epidemiología , Masculino , Persona de Mediana Edad , Fiebre por Flebótomos/virología , Phlebovirus/clasificación , Phlebovirus/genética , Phlebovirus/inmunología , ARN Viral/genética , Estudios Retrospectivos , Estaciones del Año , Adulto Joven
12.
Viruses ; 13(2)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578722

RESUMEN

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the "Europe wildlife" lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies.


Asunto(s)
Virus del Moquillo Canino/aislamiento & purificación , Moquillo/epidemiología , Moquillo/virología , Animales , Autopsia/veterinaria , Moquillo/patología , Virus del Moquillo Canino/genética , Italia/epidemiología , Proteínas de la Nucleocápside/genética , ARN Viral/genética , Estudios Retrospectivos , Vacunas Virales/genética
13.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578973

RESUMEN

A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection.


Asunto(s)
Infecciones por Coronavirus/diagnóstico , Coronavirus/aislamiento & purificación , Coronavirus/genética , Pruebas Diagnósticas de Rutina , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Pruebas en el Punto de Atención , ARN Viral/genética , Sensibilidad y Especificidad
14.
BMC Microbiol ; 21(1): 56, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33607939

RESUMEN

BACKGROUND: Gastrointestinal symptoms are common in COVID-19 patients and SARS-CoV-2 RNA has been detected in the patients' feces, which could lead to fecal-oral transmission. Therefore, fecal sample testing with real-time RT-PCR is highly recommended as a routine test for SARS-CoV-2 infection. However, varying rates of detection in fecal sample have been reported. The aim of this study was to provide insights into the detection rates of SARS-CoV-2 in COVID-19 patients' fecal sample by using four real-time RT-PCR kits and two pretreatment methods (inactive and non-inactive). RESULTS: The detection rate of Trizol pretreatment group was slightly higher than that of Phosphate Buffered Saline (PBS) groups, showing that pretreatment and inactivation by Trizol had no influence to SARS-CoV-2 nucleic acid test (NAT) results. 39.29% detection rate in fecal sample by DAAN was obtained, while Bio-germ was 40.48%, Sansure 34.52%, and GeneoDx 33.33%. The former three kits had no significant difference. The DAAN kit detection rates of ORF1ab and N gene were nearly equal and Ct value distribution was more scattered, while the Bio-germ kit distribution was more clustered. The positive rate of SARS-COV-2 in fecal samples correlated with the severity of the disease, specifically, severe cases were less likely to be identified than asymptomatic infection in the DAAN group (adjusted OR 0.05, 95%CI = 0.00 ~ 0.91). CONCLUSIONS: Trizol should be of choice as a valid and safe method for pretreatment of fecal samples of SARS-CoV-2. All real-time RT-PCR kits assessed in this study can be used for routine detection of SARS-CoV-2 in fecal samples. While DAAN, with high NAT positive rate, could be the best out of the 4 kits used in this study. SARS-CoV-2 positive rate in fecal sample was related to the severity of illness.


Asunto(s)
/diagnóstico , Heces/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /patogenicidad , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Lectura Abierta/genética , ARN Viral/genética , /aislamiento & purificación
15.
J Vis Exp ; (168)2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33616108

RESUMEN

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.


Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Impresión Tridimensional , Transcripción Reversa/genética , /aislamiento & purificación , /genética , Recursos en Salud , Humanos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos
16.
Talanta ; 225: 121978, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33592726

RESUMEN

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.


Asunto(s)
Infecciones por Caliciviridae/diagnóstico , Colorimetría/métodos , Gastroenteritis/diagnóstico , Infecciones por Caliciviridae/virología , Colorimetría/economía , Colorimetría/instrumentación , Análisis Costo-Beneficio , Gastroenteritis/virología , Genotipo , Humanos , Norovirus/genética , Norovirus/fisiología , Técnicas de Amplificación de Ácido Nucleico/métodos , Papel , ARN Viral/genética , Sensibilidad y Especificidad
17.
Talanta ; 225: 121986, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33592734

RESUMEN

Diagnostic tools play significant roles in the fight against COVID-19 and other pandemics. Existing tests, such as RT-qPCR, have limitations including long assay time, low throughput, inadequate sensitivity, and suboptimal portability. Emerging biosensing technologies hold the promise to develop tests that are rapid, highly sensitive, and suitable for point-of-care testing, which could significantly facilitate the testing of COVID-19. Despite that, practical applications of such biosensors in pandemics have yet to be achieved. In this review, we consolidate the newly developed diagnostic tools for COVID-19 using emerging biosensing technologies and discuss their application promise. In particular, we present nucleic acid tests and antibody tests of COVID-19 based on both conventional and emerging biosensing methods. We then provide perspectives on the existing challenges and potential solutions.


Asunto(s)
Técnicas Biosensibles/métodos , ARN Viral/genética , /genética , /epidemiología , /métodos , Humanos , Pandemias , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
18.
PLoS One ; 16(2): e0246173, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33529260

RESUMEN

We report clinical profile of hundred and nine patients with SARS CoV-2 infection, and whole genome sequences (WGS) of seven virus isolates from the first reported cases in India, with various international travel histories. Comorbidities such as diabetes, hypertension, and cardiovascular disease were frequently associated with severity of the disease. WBC and neutrophil counts showed an increase, while lymphocyte counts decreased in patients with severe infection suggesting a possible neutrophil mediated organ damage, while immune activity may be diminished with decrease in lymphocytes leading to disease severity. Increase in SGOT, SGPT and blood urea suggests the functional deficiencies of liver, heart, and kidney in patients who succumbed to the disease when compared to the group of recovered patients. The WGS analysis showed that these isolates were classified into two clades: I/A3i, and A2a (four according to GISAID: O, L, GR, and GH). Further, WGS phylogeny and travel history together indicate possible transmission from Middle East and Europe. Three S protein variants: Wuhan reference, D614G, and Y28H were identified predicted to possess different binding affinities to host ACE2.


Asunto(s)
/virología , Genoma Viral , Secuenciación Completa del Genoma , Adulto , Anciano , /metabolismo , /patología , Femenino , Humanos , India , Masculino , Persona de Mediana Edad , Filogenia , ARN Viral/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo
19.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33536313

RESUMEN

The characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral kinetics in hospitalized patients and its association with mortality is unknown. We analyzed death and nasopharyngeal viral kinetics in 655 hospitalized patients from the prospective French COVID cohort. The model predicted a median peak viral load that coincided with symptom onset. Patients with age ≥65 y had a smaller loss rate of infected cells, leading to a delayed median time to viral clearance occurring 16 d after symptom onset as compared to 13 d in younger patients (P < 10-4). In multivariate analysis, the risk factors associated with mortality were age ≥65 y, male gender, and presence of chronic pulmonary disease (hazard ratio [HR] > 2.0). Using a joint model, viral dynamics after hospital admission was an independent predictor of mortality (HR = 1.31, P < 10-3). Finally, we used our model to simulate the effects of effective pharmacological interventions on time to viral clearance and mortality. A treatment able to reduce viral production by 90% upon hospital admission would shorten the time to viral clearance by 2.0 and 2.9 d in patients of age <65 y and ≥65 y, respectively. Assuming that the association between viral dynamics and mortality would remain similar to that observed in our population, this could translate into a reduction of mortality from 19 to 14% in patients of age ≥65 y with risk factors. Our results show that viral dynamics is associated with mortality in hospitalized patients. Strategies aiming to reduce viral load could have an effect on mortality rate in this population.


Asunto(s)
/mortalidad , Modelos Teóricos , Nasofaringe/virología , ARN Viral/análisis , Carga Viral , Anciano , Anticuerpos Antivirales/sangre , /epidemiología , Femenino , Francia/epidemiología , Hospitalización , Humanos , Cinética , Masculino , Pronóstico , Estudios Prospectivos , ARN Viral/genética , Factores de Riesgo , Tasa de Supervivencia
20.
Euro Surveill ; 26(5)2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33541485

RESUMEN

In June-November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV-2-PCR-positive and approximately 27% (95% confidence interval (CI): 25-30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.


Asunto(s)
Animales Salvajes/virología , /veterinaria , Brotes de Enfermedades/veterinaria , Reservorios de Enfermedades/veterinaria , Transmisión de Enfermedad Infecciosa/veterinaria , Visón/virología , Pandemias/veterinaria , /aislamiento & purificación , /transmisión , Animales , /virología , Dinamarca/epidemiología , Brotes de Enfermedades/estadística & datos numéricos , Reservorios de Enfermedades/virología , Granjas , Genes Virales , Humanos , Incidencia , Reacción en Cadena de la Polimerasa , Salud Pública , ARN Viral/análisis , ARN Viral/genética , /virología , Secuenciación Completa del Genoma , Zoonosis/transmisión , Zoonosis/virología
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