Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 12.730
Filtrar
1.
J Infect Dev Ctries ; 15(2): 237-241, 2021 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-33690206

RESUMEN

INTRODUCTION: We aim to describe the performance of combined IgM and IgG point-of-care antibody test (POC-Ab) (Wondfo®) compared to real-time reverse transcriptase (rRT-PCR) (Allplex™ 2019-nCoV Assay) in detecting coronavirus disease 2019 (COVID-19). METHODOLOGY: We compared POC-Ab with rRT-PCR results among patients in a tertiary hospital from January to March 2020 in Bandung, Indonesia. We selected presumptive COVID-19 patients with positive rRT-PCR consecutively and 20 patients with negative rRT-PCR results were selected randomly from the same group of patients as controls. We described the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) with corresponding 95% confidence interval using serum and capillary blood samples. We also tested POC-Ab using non-COVID-19 (confirmed dengue and typhoid) patients' sera. RESULTS: Twenty-seven patients with positive rRT-PCR result and 20 negative controls were included (68.1% males, mean age 46 (SD: 15.4)). Using the serum, the sensitivity of the POC-Ab was 63.0% (42.4-80.6), specificity was 95.0% (75.1-99.9), PPV was 94.4% (72.7-99.8), NPV was 65.5% (45.7-82.1). A subset of 20 patients was tested using a capillary blood sample. The accuracy of the capillary blood sample is lower compared to serum (50.0% vs. 78.7%). None of the non-COVID-19 sera tested were reactive. CONCLUSIONS: POC-Ab for COVID-19 has a high specificity with no false-positive result in non-COVID-19 sera. Therefore, it can be used to guide diagnostic among symptomatic patients in resource limited settings. Given its low sensitivity, patients with high suspicion of COVID-19 but non-reactive result should be prioritized for rRT-PCR testing.


Asunto(s)
/métodos , Adulto , Anciano , /etiología , Reacciones Falso Positivas , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Indonesia , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Centros de Atención Terciaria
2.
J Infect Dev Ctries ; 15(2): 242-246, 2021 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-33690207

RESUMEN

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded.


Asunto(s)
/epidemiología , Mutación , Glicoproteína de la Espiga del Coronavirus/genética , Amplificación de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Italia/epidemiología , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación
3.
Talanta ; 228: 122227, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33773731

RESUMEN

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including open reading frame 1ab (ORF1ab), nucleocapsid protein (N) and envelope protein (E) gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Fosfodiesterasa I/química , Proteolisis , ARN/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Cromatografía Líquida de Alta Presión/normas , Crotalinae , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Nucleótidos de Purina/normas , Nucleótidos de Pirimidina/normas , ARN/química , Estándares de Referencia
4.
Mem Inst Oswaldo Cruz ; 116: e200571, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33681890

RESUMEN

Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.


Asunto(s)
Insectos Vectores/parasitología , Leishmania infantum/genética , Leishmania/aislamiento & purificación , Leishmaniasis Visceral/transmisión , Psychodidae/parasitología , Animales , Brasil , Expresión Génica , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/parasitología , Estadios del Ciclo de Vida , Psychodidae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos
5.
Methods Mol Biol ; 2292: 121-131, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33651357

RESUMEN

Bladder cancer has a very high frequency of recurrence and therefore requires close clinical surveillance throughout its life, with cystoscopies and serial cytological examinations. These tests are both invasive and expensive, with considerable interpersonal and inter-institutional variability. Moreover, cytological examination used for the diagnosis of low-grade tumors has a low sensitivity; thus, there is an increasing focus on the research for new, accurate, urinary markers. Herein, the biological basis, methodologies, and diagnostic performance of biomarkers are discussed.


Asunto(s)
Neoplasias de la Vejiga Urinaria/orina , Antígenos de Neoplasias/orina , Biomarcadores de Tumor/orina , Humanos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Proteínas Nucleares/orina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Neoplasias de la Vejiga Urinaria/diagnóstico
6.
Dis Markers ; 2021: 8890221, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33747257

RESUMEN

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. Methods: The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). Results: The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. Conclusion: During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.


Asunto(s)
/métodos , Nasofaringe/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /genética , Portador Sano/virología , Humanos , Límite de Detección , ARN Viral/genética , Carga Viral , Flujo de Trabajo
7.
J Coll Physicians Surg Pak ; 30(1): S1-S6, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33650414

RESUMEN

OBJECTIVE: To compare the diagnostic accuracies of HRCT chest and RT-PCR results in diagnosis of coronavirus disease (COVID-19) in a tertiary care hospital in Lahore. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Department of Radiology and Central Research Lab, Lahore General Hospital, Lahore, from April to July, 2020. METHODOLOGY: Patients aged 18 to 83 years, who had clinically suspected symptoms of COVID-19 (fever, cough/sore throat or shortness of breath) presenting in outpatient or emergency department, were included. These patients had their HRCT chest conducted from Radiology Department and RT-PCR performed at Central Research Lab. These data were retrieved from electronic system of PACS. Results were categorised into positive and negative findings for COVID-19. Diagnostic accuracies of HRCT chest and first RT-PCR along with 95% confidence interval were calculated. RESULTS: A total of 94 patients, 55 (58.5%) males and 39 (41.5%) were females. Out of them, 83% patients had positive HRCT chest findings of COVID-19, 17% had negative HRCT chest findings; while 40.4% had positive and 59.6% had negative first PCR. Among the repeat second PCR, 19.6% had negative, 1.8% had positive PCR results; while 78.6% patients didn't undergo repeat PCR. The sensitivity, specificity, NPV, PPV and accuracy of HRCT chest was 92%, 23%, 81%, 45%, and 51%; while of first RT-PCR was 45%, 81%, 23%, 92% and 51%, respectively. CONCLUSION: The sensitivity of HRCT chest is higher (92%) as compared to first RT-PCR (45%). Key Words: COVID-19, RT-PCR, HRCT chest, Sensitivity, Specificity.


Asunto(s)
/diagnóstico , Pandemias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tomografía Computarizada por Rayos X/métodos , Adulto , Anciano , /virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pakistán/epidemiología , Estudios Retrospectivos
8.
J Coll Physicians Surg Pak ; 30(1): S26-S28, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33650420

RESUMEN

We present a case of coronavirus disease 2019 (COVID-19) re-infection where the time interval between two COVID-positive episodes is the longest in the literature. A 40-year male patient was admitted to the Emergency Department with  complaints of sore throat, cough and diarrhea; and was re-diagnosed as COVID-19 positive after a virus-free period. He did not have a chronic disease in his anamnesis and used no medication. After COVID-19 infection and a long recovery period, he became COVID-19 positive again. In this case, the time to second COVID-19 infection was 94 days from the first positive PCR test and 86 days from the complete resolution of symptoms. This is one of the longest COVID-19-free period between two episodes of infection in the literature. Key Words: COVID-19, Recurrence, Re-infection, Recovery.


Asunto(s)
/epidemiología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /genética , Adulto , /virología , Humanos , Masculino , Pandemias , Recurrencia
9.
J Infect Dis ; 223(2): 206-213, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33535237

RESUMEN

BACKGROUND: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. METHODS: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. RESULTS: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. CONCLUSIONS: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.


Asunto(s)
/métodos , /virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Pruebas en el Punto de Atención , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , /genética
10.
J Virol Methods ; 291: 114086, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33577957

RESUMEN

The worldwide demand for SARS-CoV-2 RT-PCR testing resulted in a shortage of diagnostic kits. RNA extraction step constitutes a major bottleneck to perform diagnostic. The aim of this study was to assess performances of different extraction-free SARS-CoV-2 RT-PCR assays compared to a reference RT-PCR assay. The panel of evaluation consisted of 94 samples: 69 positive and 25 negative for SARS-CoV-2 by reference RT-PCR. Three extraction-free RT-PCR assays were assessed: (i) PrimeDirect® Probe RT-qPCR Mix (Takara), (ii) PrimeScript®RT-PCR (Takara), and (iii) SARS-CoV-2 SANSURE®BIOTECH Novel Coronavirus (Sansure). The overall sensitivity of PrimeDirect, PrimeScript and Sansure assays was 55.1 %, 69.6 % and 69.6 %, respectively. The sensitivity increased among samples with Ct<30: 91.9 % (n = 34/37), 89.2 % (n = 33/37) and 94.6 % (n = 35/37) for PrimeDirect, PrimeScript and Sansure assays, respectively. The specificity was 88 %, 100 % and 100 % for PrimeDirect, PrimeScript and Sansure assays, respectively. In the present study, we showed a good sensitivity of extraction-free PCR assays, especially for high viral loads (Ct<30), except PrimeDirect that displayed imperfect sensitivity and specificity. Despite a lower sensitivity for low viral loads, extraction-free reagents can provide a valuable option, cheaper, easier and less reagent consuming for SARS-CoV-2 diagnostic, especially in laboratory with lower experience and equipment for molecular assays.


Asunto(s)
/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación , /virología , Técnicas de Laboratorio Clínico/métodos , ARN Viral/genética , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Carga Viral
11.
J Int Med Res ; 49(2): 300060520972658, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33530774

RESUMEN

BACKGROUND: In December 2019, an outbreak of coronavirus disease 2019 (COVID-19) began in Wuhan, China, and led to a global epidemic. We aimed to compare the clinical and serological features of COVID-19 patients with positive and negative reverse transcriptase polymerase chain reaction (RT-PCR) tests. METHODS: This was a retrospective cohort study conducted from 9 February to 4 April 2020. COVID-19 patients at Leishenshan Hospital in Wuhan, China (125 total cases; 87 RT-PCR positive and 38 RT-PCR negative) were included. COVID-19 serology was assessed by colloidal gold assay. All cases were analyzed for demographic, clinical, and serological features. RESULTS: There were no significant differences in most demographic features, clinical symptoms, complications or treatments of RT-PCR positive and negative COVID-19 patients. Serum IgM/IgG was positive in 82 (94%) and 33 (87%) RT-PCR positive and negative cases, respectively. IgM was detectable as early as 3 days after symptom onset and was undetectable 60 days after symptom onset. By contrast, IgG could be detected only 10 days after symptom onset and reached its peak 60 days after symptom onset. CONCLUSIONS: Serological tests performed during the appropriate time window of disease progression could be valuable auxiliary methods to RT-PCR in COVID-19 patients.


Asunto(s)
/patología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos
12.
Pathog Dis ; 79(1)2021 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-33537743

RESUMEN

BACKGROUND: despite being in the 5th month of pandemic, knowledge with respect to viral dynamics, infectivity and RT-PCR positivity continues to evolve. AIM: to analyse the SARS CoV-2 nucleic acid RT-PCR profiles in COVID-19 patients. DESIGN: it was a retrospective, observational study conducted at COVID facilities under AIIMS, New Delhi. METHODS: patients admitted with laboratory confirmed COVID-19 were eligible for enrolment. Patients with incomplete details, or only single PCR tests were excluded. Data regarding demographic details, comorbidities, treatment received and results of SARS-CoV-2 RT-PCR performed on nasopharyngeal and oropharyngeal swabs, collected at different time points, was retrieved from the hospital records. RESULTS: a total of 298 patients were included, majority were males (75·8%) with mean age of 39·07 years (0·6-88 years). The mean duration from symptom onset to first positive RT-PCR was 4·7 days (SD 3·67), while that of symptom onset to last positive test was 17·83 days (SD 6·22). Proportions of positive RT-PCR tests were 100%, 49%, 24%, 8·7% and 20·6% in the 1st, 2nd, 3rd, 4th and >4 weeks of illness. A total of 12 symptomatic patients had prolonged positive test results even after 3 weeks of symptom onset. Age > = 60 years was associated with prolonged RT-PCR positivity (statistically significant). CONCLUSION: this study showed that the average period of PCR positivity is more than 2 weeks in COVID-19 patients; elderly patients have prolonged duration of RT-PCR positivity and requires further follow up.


Asunto(s)
/virología , /genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , /métodos , Niño , Preescolar , Comorbilidad , Femenino , Hospitalización , Humanos , India/epidemiología , Lactante , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Orofaringe/virología , Pandemias , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adulto Joven
13.
Methods Mol Biol ; 2273: 219-238, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604857

RESUMEN

Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.


Asunto(s)
Bovinos/genética , Vesículas Extracelulares/genética , Trompas Uterinas/metabolismo , MicroARNs/genética , Animales , Western Blotting/métodos , Cromatografía en Gel/métodos , Femenino , Perfilación de la Expresión Génica/métodos , MicroARNs/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transcriptoma , Ultracentrifugación/métodos , Útero/metabolismo
14.
PLoS One ; 16(2): e0243183, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33621263

RESUMEN

BACKGROUND: Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling. METHODS: Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated. RESULTS: The LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100-200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). CONCLUSION: The studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.


Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Saliva/virología , Manejo de Especímenes/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Adulto Joven
15.
Talanta ; 225: 121898, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33592692

RESUMEN

The current situation of the Covid-19 pandemic is indicated by a huge number of infections, high lethality, and rapid spread. These circumstances have stopped the activity of almost the entire world, affecting severely the global economy. A rapid diagnosis of the Covid-19 and a generalized testing protocol is essential to fight against the pandemic and to maintain health control in the population. Principal biosensing and diagnostic technologies used to monitor the spread of the SARS-CoV-2 are based on specific genomic analysis and rapid immune tests, both with different technology platforms that include advantages and disadvantages. Most of the in vitro diagnosis companies are competing to be the first on validating under different regulations their technology for placing their platforms for Covid-19 detection as fast as possible in this big international market. A comprehensive analysis of the commercialized technologies for the genomic based sensing and the antibody/antigen detection methods devoted to Covid-19 diagnosis is described in this review, which have been detailed and listed under different countries regulations. The effectiveness of the described technologies throughout the different stages of the disease and a critical comparison of the emerging technologies in the market to counterattack this pandemic have been discussed.


Asunto(s)
/diagnóstico , Inmunoensayo/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , /virología , Humanos , Pandemias , /fisiología , Sensibilidad y Especificidad
16.
Mem Inst Oswaldo Cruz ; 115: e200287, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33533869

RESUMEN

BACKGROUND: The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES: To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS: Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS: Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS: Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.


Asunto(s)
Antígenos Virales/sangre , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Inmunológicas/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas no Estructurales Virales/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Dengue/sangre , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/inmunología , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/genética
17.
Indian J Med Microbiol ; 39(1): 116-117, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33610241

RESUMEN

There are several reports of Ct values of RT PCR assays for COVID 19 being associated with disease severity and infectivity. We studied the correlation between Ct values and disease severity and mortality at our hospital . All patients with RT PCR diagnosed COVID 19 illness admitted at the study site and for whom Ct values were available were included in the study. The patients with mild disease had significantly lower Ct values than patients with severe disease but had also been tested significantly earlier in the illness than those with severe disease. The patients who died had significantly lower Ct values than patients who survived but here again they had significantly shorter duration of symptoms before testing. We therefore recommend that the time of testing since onset of symptoms should be controlled for while correlating Ct values with disease severity.


Asunto(s)
/mortalidad , /virología , /diagnóstico , Femenino , Humanos , Masculino , Mortalidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /genética , Índice de Severidad de la Enfermedad
18.
Indian J Med Microbiol ; 39(1): 133-135, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33610247

RESUMEN

In the modern COVID-19 pandemic, reverse transcription-polymerase chain reaction (RT-PCR) positivity has a major role in the diagnosis of the disease. However, in deciding the patient's discharge or de-isolation, its role is still debatable. We are, hereby, describing three cases (an intern, a nursing officer and a caretaker of another patient) where only RT-PCR could not help much since it was persistently positive for >20 days of the illness course. Instead, the cycle threshold (Ct) values could have better correlated with the infectivity of COVID. We propose a rising trend (24 h apart) and absolute Ct value > 25, instead of RT-PCR negativity (which was taken as Ct value > 36 in our laboratory), to be used in deciding the infective potential of the patients, their discharge from the hospital and de-isolation of the patients. This will help in the timely discharge of patients from health-care institutions and home isolation, which, as a result, will lead to optimal utilisation of the limited hospital resources we have available in the line of the ongoing pandemic. Future studies are required to define the exact cut-off of Ct value for de-isolation purposes.


Asunto(s)
/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , /genética , Adulto , Manejo de la Enfermedad , Femenino , Hospitalización , Humanos , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Índice de Severidad de la Enfermedad , Evaluación de Síntomas , Adulto Joven
19.
J Clin Virol ; 136: 104764, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33636553

RESUMEN

The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 °C for 30 min, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Enfermedades Asintomáticas , Canadá , Humanos , Tamizaje Masivo/métodos , Nasofaringe/virología , ARN Viral/análisis , Sensibilidad y Especificidad
20.
Sci Rep ; 11(1): 2388, 2021 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-33504923

RESUMEN

Reverse transcriptase-polymerase chain reaction (RT-PCR) testing is an important tool for diagnosing coronavirus disease 2019 (COVID-19). However, performance concerns have emerged recently, notably regarding sensitivity. We hypothesized that the clinical, biological, and radiological characteristics of patients with a false-negative first RT-PCR test and a final diagnosis of COVID-19 might differ from those of patients with a positive first RT-PCR test. We conducted a multicenter matched case-control study in COVID-19 patients. Patients with a negative first RT-PCR test were matched to patients with a positive first RT-PCR test on age, sex, and initial admission unit (ward or intensive care). We included 80 cases and 80 controls between March 30, and June 22, 2020. Neither mortality at hospital discharge nor hospital stay length differed between the two groups (P = 0.80 and P = 0.54, respectively). By multivariate analysis, two factors were independently associated with a lower risk of a first false-negative test, namely, headache (adjusted OR [aOR], 0.07; 95% confidence interval [95% CI], 0.01-0.49]; P = 0.007) and fatigue/malaise (aOR, 0.16; 95% CI, 0.03-0.81; P = 0.027); two other factors were independently associated with a higher risk of a first false-negative test, namely, platelets > 207·103 mm-3 (aOR, 3.81; 95% CI, 1.10-13.16]; P = 0.034) and C-reactive protein > 79.8 mg·L-1 (aOR, 4.00; 95% CI, 1.21-13.19; P = 0.023). Patients with suspected COVID-19 whose laboratory tests indicating marked inflammation were at higher risk of a first false-negative RT-PCR test. Strategies involving serial RT-PCR testing must be rigorously evaluated.


Asunto(s)
/métodos , /virología , /genética , Adulto , Anciano , /estadística & datos numéricos , Estudios de Casos y Controles , Reacciones Falso Negativas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...