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1.
PLoS One ; 16(2): e0246544, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33539474

RESUMEN

To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.


Asunto(s)
/métodos , /genética , Manejo de Especímenes/métodos , /virología , Humanos , Tamizaje Masivo/métodos , Nasofaringe/virología , ARN Viral/análisis , ARN Viral/genética , Estudios Retrospectivos , /aislamiento & purificación
2.
Tohoku J Exp Med ; 253(2): 101-108, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33536379

RESUMEN

In response to the COVID-19 pandemic caused by SARS-CoV-2 in 2020, we conducted drive-through nasopharyngeal swab testing for COVID-19 in Sendai city, Japan, since April 2020. All tested individuals were judged in advance by public health centers for the necessity of undergoing the test with possible contact history and/or symptoms suggestive of COVID-19. In this study, to identify the predictors of SARS-CoV-2 test positivity for more efficient and evidenced selection of suspected individuals, we enrolled 3,540 consecutive individuals, tested in the first 7 months of the testing program, with data regarding to the history of close contact with COVID-19 patients, including those involved in cluster outbreaks. This cohort included 284 foreign students (257 males and 27 females) from a vocational school involved in the largest cluster outbreak in the area. Close contact history was present in 952 (26.9%) of the participants. The reverse transcription-polymerase chain reaction (RT-PCR) test results showed that 164 participants (4.6%) were positive and 3,376 participants (95.4%) were negative for the SARS-CoV-2 nucleocapsid gene (N2). In the univariate and multivariate analyses, history of close contact with COVID-19 patients, higher age, cough symptoms, and non-native ethnicity were predictors for SARS-CoV-2 test positivity. However, the significance of age and foreign nationality disappeared or declined upon excluding the foreign students from the aforementioned largest cluster outbreak. In conclusion, a history of close contact with COVID-19 patients and the presence of cough symptoms are significant predictors of SARS-CoV-2 test positivity.


Asunto(s)
/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Manejo de Especímenes/métodos , Adulto , Brotes de Enfermedades , Femenino , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Desarrollo de Programa , Salud Pública , Adulto Joven
3.
Sci Rep ; 11(1): 3122, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542424

RESUMEN

Sample pooling strategy was intended to determine the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2 and process them without significant loss of test usability. Standard molecular diagnostic laboratory equipment, and commercially available centrifugal filters, RNA isolation kits and SARS Cov2 PCR tests were used. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single extraction column. Out of 16 tested pools, 12 were positive with cycle threshold (Ct) values within 0.5 and 3.01 Ct of the original individual specimens. The analysis of 112 specimens determined that 12 pools were positive, followed by identification of 6 positive individual specimens among the 112 tested. This testing was accomplished with the use of 16 extractions/PCR tests, resulting in saving of 96 reactions but adding the 40 centrifugal filters. The present study demonstrated that pool testing could detect even up to a single positive sample with Ct value as high as 34. According to the standard protocols, reagents and equipment, this pooling method can be applied easily in current clinical testing laboratories.


Asunto(s)
/métodos , ARN Viral/aislamiento & purificación , Manejo de Especímenes/métodos , Humanos , /aislamiento & purificación , Sensibilidad y Especificidad
4.
Popul Health Manag ; 24(S1): S26-S34, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33544647

RESUMEN

Laboratory testing is an important component in the diagnosis of respiratory tract infections such as with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, specimen collection not only risks exposure of health care workers and other patients to infection, but also necessitates use of personal protective equipment that may be in short supply during periods of heightened disease activity. Self-collection of nasal or oropharyngeal swabs offers an alternative to address these drawbacks. Although studies in the past decade have demonstrated the utility of this approach for respiratory infections, it has not been widely adopted in routine clinical practice. The rapid spread of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has focused attention on the need for safe, convenient, timely, and scalable methods for collecting upper respiratory specimens for testing. The goals of this article are to highlight the literature regarding self-collected nasal or oropharyngeal specimens for respiratory pathogen testing; discuss the role of self-collection in helping prevent the spread of the COVID-19 disease from infected patients and facilitating a shift toward "virtual" medicine or telemedicine; and describe the current and future state of self-collection for infectious agents, and the impacts these approaches can have on population health management and disease diagnosis and prevention.


Asunto(s)
Gestión de la Salud Poblacional , Manejo de Especímenes/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , /prevención & control , Niño , Preescolar , Humanos , Lactante , Persona de Mediana Edad , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/virología , Autocuidado , Telemedicina , Adulto Joven
5.
J Vis Exp ; (167)2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33554968

RESUMEN

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.


Asunto(s)
Abejas/fisiología , Polen/fisiología , Manejo de Especímenes/métodos , Ácido Acético/química , Animales , Polinización , Coloración y Etiquetado
6.
JMIR Public Health Surveill ; 7(1): e24220, 2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33406478

RESUMEN

BACKGROUND: Real-time polymerase chain reaction using nasopharyngeal swabs is currently the most widely used diagnostic test for SARS-CoV-2 detection. However, false negatives and the sensitivity of this mode of testing have posed challenges in the accurate estimation of the prevalence of SARS-CoV-2 infection rates. OBJECTIVE: The purpose of this study was to evaluate whether technical and, therefore, correctable errors were being made with regard to nasopharyngeal swab procedures. METHODS: We searched a web-based video database (YouTube) for videos demonstrating SARS-CoV-2 nasopharyngeal swab tests, posted from January 1 to May 15, 2020. Videos were rated by 3 blinded rhinologists for accuracy of swab angle and depth. The overall score for swab angle and swab depth for each nasopharyngeal swab demonstration video was determined based on the majority score with agreement between at least 2 of the 3 reviewers. We then comparatively evaluated video data collected from YouTube videos demonstrating the correct nasopharyngeal swab technique with data from videos demonstrating an incorrect nasopharyngeal swab technique. Multiple linear regression analysis with statistical significance set at P=.05 was performed to determine video data variables associated with the correct nasopharyngeal swab technique. RESULTS: In all, 126 videos met the study inclusion and exclusion criteria. Of these, 52.3% (66/126) of all videos demonstrated the correct swab angle, and 46% (58/126) of the videos demonstrated an appropriate swab depth. Moreover, 45.2% (57/126) of the videos demonstrated both correct nasopharyngeal swab angle and appropriate depth, whereas 46.8% (59/126) of the videos demonstrated both incorrect nasopharyngeal swab angle and inappropriate depth. Videos with correct nasopharyngeal swab technique were associated with the swab operators identifying themselves as a medical professional or as an Ear, Nose, Throat-related medical professional. We also found an association between correct nasopharyngeal swab techniques and recency of video publication date (relative to May 15, 2020). CONCLUSIONS: Our findings show that over half of the videos documenting the nasopharyngeal swab test showed an incorrect technique, which could elevate false-negative test rates. Therefore, greater attention needs to be provided toward educating frontline health care workers who routinely perform nasopharyngeal swab procedures.


Asunto(s)
/métodos , Nasofaringe/virología , Medios de Comunicación Sociales , Manejo de Especímenes/métodos , Grabación en Video , Errores Diagnósticos/prevención & control , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa
7.
J Nepal Health Res Counc ; 18(4): 785-788, 2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33510529

RESUMEN

Mobile Swab Collection Vehicle is a concept that has been innovated by a team of medical and non-medical personals based upon the dire need to perform throat or nasopharyngeal swab collections to do reverse transcription polymerase chain reaction  'RT-PCR' quickly, safely, and comfortably during this Corona Virus Diseases 2019, 'COVID-19' pandemic in a resource-poor country like Nepal. Inputs from the health care workers, beneficiaries, and engineers have been taken to construct this vehicle-mounted booth for swab collection. This vehicle is the 'first of its kind' in Nepal. Keywords: COVID-19; Nepal; swab collection vehicle.


Asunto(s)
/métodos , Unidades Móviles de Salud/organización & administración , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Manejo de Especímenes/métodos , Humanos , Nepal
8.
J Proteome Res ; 20(2): 1434-1443, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33497234

RESUMEN

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.


Asunto(s)
/diagnóstico , Cromatografía Liquida/métodos , Nasofaringe/virología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Espectrometría de Masas en Tándem/métodos , /virología , Medios de Cultivo , Humanos , Nucleocápside/metabolismo , Proteómica/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Manejo de Especímenes/instrumentación , Manejo de Especímenes/métodos , Proteínas Virales/metabolismo
9.
J Clin Virol ; 135: 104720, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33418142

RESUMEN

BACKGROUND: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction. OBJECTIVE: Multicenter evaluation of the QIAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods. STUDY DESIGN: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data. RESULTS: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of CT values, showed almost complete concordance in the QIA P&A assay for samples up to CT values of 33 with one exception of CT 31. Prospectively 60 samples were tested and also showed 100 % concordance with 5 positives. The method has been automated by two centres. CONCLUSIONS: Despite an input of only 8 µL of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic.


Asunto(s)
/métodos , /virología , Tamizaje Masivo/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /genética , Técnicas de Laboratorio Clínico/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Pandemias/prevención & control , Estudios Prospectivos , Manejo de Especímenes/métodos , Flujo de Trabajo
10.
Respiration ; 100(1): 27-33, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33412544

RESUMEN

BACKGROUND: Bronchoscopic sampling of bronchoalveolar fluid (BAL) should be safe and effective. Current sampling practice risks loss of sample to the attached negative flow, aerosolisation, or spillage, due to repeated circuit breaks, when replacing sample containers. Such concerns were highlighted during the recent coronavirus pandemic. OBJECTIVES: Evaluation of an alternative integrated sampling solution, with the Ambu Bronchosampler with aScope 4, by an experienced bronchoscopist in ICU. METHODS: An observational study of 20 sequential bronchoscopic diagnostic sampling procedures was performed on mechanically ventilated patients with suspected ventilator-associated pneumonia. Mixed methods assessment was done. The predefined outcome measures were (1) ease of set up, (2) ease of specimen collection, (3) ease of protecting specimen from loss or spillage, and (4) overall workflow. The duration of the procedure and the % volume of sample retrieved were recorded. RESULTS: The mean (±standard deviation [SD]) time for collecting 1 sample was 2.5 ± 0.8 min. The mean (±SD) specimen yield for instilled miniBAL was 54.2 ± 17.9%. Compared with standard sampling, the set-up was much easier in 18 (90%), or easier in 2 (10%) of procedures, reducing the connection steps. It was much more intuitive to use in 14 (70%), more intuitive in 4 (20%), and no more intuitive to use in 2 (10%). The overall set-up and workflow was much easier in 69% of the 13 intraprocedural connections and easier or as easy in the remaining 31% procedures. All procedures where pre connection was established were much easier (7, 100%). The Ambu Bronchosampler remained upright in all procedures with no loss or spillage of sample. Obtaining a sample was much easier in 60%, easier in 10%, no different in 20%, and worse in 10%. The ability to protect a sample from start to finish compared to standard procedures was much easier in 80%, easier in 15%, and no different in 5% of procedures. Overall workflow was much easier in 14 (70%), easier in 4 (20%), and no different in 2 (10%) of procedures. CONCLUSIONS: The Ambu Bronchosampler unit was a reliable, effective, and possibly safer technique for diagnostic sampling in ICU. It may improve safety standards during the coronavirus pandemic. A randomized control trial against the standard sampling technique is warranted.


Asunto(s)
Broncoscopios , Broncoscopía/métodos , Equipos Desechables , Respiración Artificial , Manejo de Especímenes/métodos , Lavado Broncoalveolar/instrumentación , Lavado Broncoalveolar/métodos , Líquido del Lavado Bronquioalveolar , Broncoscopía/instrumentación , /transmisión , Humanos , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/prevención & control , Exposición Profesional/prevención & control , Aisladores de Pacientes , Equipo de Protección Personal , Neumonía Asociada al Ventilador/diagnóstico , Medición de Riesgo
11.
Nano Lett ; 21(3): 1508-1516, 2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33501831

RESUMEN

Following the COVID-19 outbreak, swabs for biological specimen collection were thrust to the forefront of healthcare materials. Swab sample collection and recovery are vital for reducing false negative diagnostic tests, early detection of pathogens, and harvesting DNA from limited biological samples. In this study, we report a new class of nanofiber swabs tipped with hierarchical 3D nanofiber objects produced by expanding electrospun membranes with a solids-of-revolution-inspired gas foaming technique. Nanofiber swabs significantly improve absorption and release of proteins, cells, bacteria, DNA, and viruses from solutions and surfaces. Implementation of nanofiber swabs in SARS-CoV-2 detection reduces the false negative rates at two viral concentrations and identifies SARS-CoV-2 at a 10× lower viral concentration compared to flocked and cotton swabs. The nanofiber swabs show great promise in improving test sensitivity, potentially leading to timely and accurate diagnosis of many diseases.


Asunto(s)
/instrumentación , Nanofibras , /virología , /estadística & datos numéricos , Reacciones Falso Negativas , Humanos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Nanofibras/ultraestructura , Nanotecnología , Manejo de Especímenes/instrumentación , Manejo de Especímenes/métodos , Manejo de Especímenes/estadística & datos numéricos
12.
J Virol Methods ; 289: 114062, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33428990

RESUMEN

BACKGROUND: Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. OBJECTIVES: To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. STUDY DESIGN: IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. RESULTS: In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08-6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. CONCLUSIONS: Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.


Asunto(s)
/métodos , ARN Viral/aislamiento & purificación , Manejo de Especímenes/métodos , Detergentes/química , Humanos
13.
BMC Microbiol ; 21(1): 31, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33482729

RESUMEN

BACKGROUND: Early 2020, a COVID-19 epidemic became a public health emergency of international concern. To address this pandemic broad testing with an easy, comfortable and reliable testing method is of utmost concern. Nasopharyngeal (NP) swab sampling is the reference method though hampered by international supply shortages. A new oropharyngeal/nasal (OP/N) sampling method was investigated using the more readily available throat swab. RESULTS: 35 patients were diagnosed with SARS-CoV-2 by means of either NP or OP/N sampling. The paired swabs were both positive in 31 patients. The one patient who tested negative on both NP and OP/N swab on admission, was ultimately diagnosed on bronchoalveolar lavage fluid. A strong correlation was found between the viral RNA loads of the paired swabs (r = 0.76; P < 0.05). The sensitivity of NP and OP/N analysis in hospitalized patients (n = 28) was 89.3% and 92.7% respectively. CONCLUSIONS: This study demonstrates equivalence of NP and OP/N sampling for detection of SARS-CoV-2 by means of rRT-PCR. Sensitivity of both NP and OP/N sampling is very high in hospitalized patients.


Asunto(s)
/diagnóstico , Pandemias , Manejo de Especímenes/métodos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Orofaringe/virología , Estudios Prospectivos , Sensibilidad y Especificidad , Adulto Joven
14.
BMJ Open ; 11(1): e043925, 2021 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-33455939

RESUMEN

OBJECTIVE: Although widespread testing for SARS-CoV-2 is in place, little is known about how well the public understands these results. We aimed to provide a comprehensive overview of the general public's grasp of the accuracy and significance of the results of the swab test. DESIGN: Web-based behavioural experiment. SETTING: Italy during the April 2020 lockdown. PARTICIPANTS: 566 Italian residents. MAIN OUTCOME MEASURES: Participants' estimates of the SARS-CoV-2 prevalence; the predictive and diagnostic accuracy of the test; the behavioural impact of (positive vs negative) test results; the perceived usefulness of a short-term repetition of the test following positive or negative results; and rankings of causes for false positives and false negatives. RESULTS: Most participants considered the swab test useful (89.6%) and provided predictive values consistent with their estimates of test diagnostic accuracy and infection prevalence (67.0%). Participants acknowledged the effects of symptomatic status and geographical location on prevalence (all p<0.001) but failed to take this information into account when estimating the positive or negative predictive value. Overall, test specificity was underestimated (91.5%, 95% CI 90.2% to 92.8%); test sensitivity was overestimated (89.7%, 95% CI 88.3% to 91.0%). Positive results were evaluated as more informative than negative ones (91.6, 95% CI 90.2 to 93.1 and 41.0, 95% CI 37.9 to 44.0, respectively, p<0.001); a short-term repetition of the test was considered more useful after a positive than a negative result (62.7, 95% CI 59.6 to 65.7 and 47.2, 95% CI 44.4 to 50.0, respectively, p=0.013). Human error and technical characteristics were assessed as more likely to be the causes of false positives (p<0.001); the level of the viral load as the cause of false negatives (p<0.001). CONCLUSIONS: While some aspects of the swab for SARS-CoV-2 are well grasped, others are not and may have a strong bearing on the general public's health and well-being. The obtained findings provide policymakers with a detailed picture that can guide the design and implementation of interventions for improving efficient communication with the general public as well as adherence to precautionary behaviour.


Asunto(s)
/diagnóstico , Técnicas de Laboratorio Clínico/métodos , Nasofaringe/virología , /genética , Adolescente , Adulto , Anciano , Reacciones Falso Negativas , Reacciones Falso Positivas , Femenino , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Prevalencia , Aislamiento Social , Manejo de Especímenes/métodos , Adulto Joven
15.
Microbiome ; 9(1): 25, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33482920

RESUMEN

BACKGROUND: Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, synthetic, and foam) and one clinical-grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients including COVID-19+ patients. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. RESULTS: Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4× higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic (SYN) swab. The limit of detection (LoD) of SARS-CoV-2 from floor samples collected using the consumer-grade plastic (CGp) or research-grade plastic The Microsetta Initiative (TMI) swabs was similar or better than the SYN swab, further suggesting that swab type does not impact RNA recovery as measured by the abundance of SARS-CoV-2. The LoD for TMI was between 0 and 362.5 viral particles, while SYN and CGp were both between 725 and 1450 particles. Lastly microbiome analyses (16S rRNA gene sequencing) of paired samples (nasal and floor from same patient room) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type, but instead driven by the patient and sample type. CONCLUSIONS: Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer-grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity and lack of antibiotics in these samples makes it possible to perform concomitant microbiome analyses. Video abstract.


Asunto(s)
/instrumentación , Microbiota , ARN Viral/análisis , Manejo de Especímenes/métodos , Transporte Biológico , Etanol/química , Estudios de Factibilidad , Humanos , Unidades de Cuidados Intensivos , Límite de Detección , ARN Ribosómico 16S/genética , ARN Viral/genética , Ribonucleasas/metabolismo
16.
J Virol Methods ; 287: 113969, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32918932

RESUMEN

The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evaluated the ability of each kit to detect viral RNA from both kit-extracted or directly from a pre-boiled NSS observing that direct RNA detection is possible when Ct values are lower than 30 with the three kits tested. Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.


Asunto(s)
/métodos , /aislamiento & purificación , Manejo de Especímenes/métodos , /virología , Humanos , Nasofaringe/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Factores de Tiempo , Carga Viral
17.
Diagn Microbiol Infect Dis ; 99(1): 115206, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33045499

RESUMEN

The diagnosis of coronavirus disease-19 (COVID-19) relies on the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA by real-time reverse-transcription polymerase chain reaction in respiratory samples. Rapid increase in the COVID-19 cases across the world requires fast and efficient testing as testing capacity is a bottleneck in diagnosis. In this context, pooling strategy can be opted for rapid testing in a cost-effective manner. In this study, the authors have optimized and compared the effect of pooling (5 and 10 samples) before and after nucleic acid extraction. It was concluded that there was no significant difference in the SARS CoV-2 RNA detection in the pools prepared at sample or RNA level. Even after pooling, 10-fold dilution was detectable with 3-cycle threshold value change in both type of pools when compared with individual samples. Hence, sample pool size of 10 can be used in low-prevalent areas, and testing capacity can be substantially increased.


Asunto(s)
/métodos , /aislamiento & purificación , Manejo de Especímenes/métodos , /normas , Genes Virales/genética , Humanos , India/epidemiología , Nasofaringe/virología , Faringe/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Manejo de Especímenes/normas , Centros de Atención Terciaria
18.
J Infect Chemother ; 27(1): 126-129, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33060046

RESUMEN

Considering the issues of shortage of medical resources and the invasiveness and infection risk involved in the collection of nasopharyngeal swab specimens, there is a need for an effective alternative test specimen for SARS-CoV-2 RNA detection. Here, we investigated suitability of saliva as a non-invasively obtained specimen for molecular detection of SARS-CoV-2 RNA in Japanese patients with COVID-19. In total, 28 paired clinical specimens of saliva and nasopharyngeal swabs were collected from 12 patients at various time points after symptom onset. Each specimen was assayed using reverse transcription real-time polymerase chain reaction (rRT-PCR) on the BD MAX open system using primers and probes targeting the N-gene. The saliva and nasopharyngeal swab specimens showed 19 and 15 positive results, respectively. No invalid (PCR inhibition) result was observed for any specimen. The qualitative results of each specimen obtained in the period immediately after symptom onset were similar. Three convalescent patients presented saliva-positive results, whereas their nasopharyngeal swabs were negative at four different time points, suggesting that saliva may be superior to nasopharyngeal swabs in terms of obtaining stable assay result of SARS-CoV-2. In conclusion, our results suggest that saliva can potentially serve as an alternative to nasopharyngeal swabs as a specimen for SARS-CoV-2 rRT-PCR. As saliva can be collected by patients themselves, it may be an effective way to overcome the shortage of personal protective equipment and specimen sampling tools.


Asunto(s)
Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/diagnóstico , Nasofaringe/virología , Neumonía Viral/diagnóstico , ARN Viral/aislamiento & purificación , Saliva/virología , Técnicas de Laboratorio Clínico , Humanos , Japón , Pandemias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Manejo de Especímenes/métodos
19.
Diagn Microbiol Infect Dis ; 99(1): 115209, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33080426

RESUMEN

Nasopharyngeal flocked swabs placed in viral transport media (VTM) are the preferred collection methodology for respiratory virus testing. Due to the rapid depletion of available reagents and swabs, we have validated an alternative swab placed in phosphate-buffered saline (PBS) for use in respiratory virus testing in a SARS-CoV-2 real-time polymerase chain reaction assay and a multiplexed respiratory virus panel. We collected nasopharyngeal (NP) swabs and oropharyngeal (OP) swabs from 10 healthy volunteers. Flocked swabs were placed in VTM and alternative swabs in PBS. In this feasibility study, we show that NP collection is better for detection of human material than OP collection, as measured by significantly lower RNase P gene cycle threshold values, and that a Dacron polyester swab in PBS shows equivalent detection of SARS-CoV-2 and RSV to a flocked swab in VTM in contrived specimens. Diluted SARS-CoV-2-positive patient specimens are detectable for up to 72 h at 4 °C.


Asunto(s)
/diagnóstico , Manejo de Especímenes/métodos , Medios de Cultivo , Estudios de Factibilidad , Humanos , Nasofaringe/virología , Orofaringe/virología , Tereftalatos Polietilenos , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/aislamiento & purificación , /genética
20.
Diagn Microbiol Infect Dis ; 99(1): 115205, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33190092

RESUMEN

Pooling of 1 positive sample with up to 5 negative samples prior to testing with the Cepheid GenXpert SARS-CoV-2 assay did not adversely impact detection of positive samples. At our current prevalence of 2%, it could save up to 70% of the test kits.


Asunto(s)
/métodos , /aislamiento & purificación , Manejo de Especímenes/métodos , Humanos , Juego de Reactivos para Diagnóstico/economía , Juego de Reactivos para Diagnóstico/provisión & distribución , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
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