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1.
PLoS One ; 16(6): e0252507, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34061896

RESUMEN

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Asunto(s)
Prueba de COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiología , Pruebas Diagnósticas de Rutina/normas , Indicadores y Reactivos/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , SARS-CoV-2/genética , COVID-19/virología , Prueba de COVID-19/métodos , Camerún/epidemiología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Ghana/epidemiología , Humanos , Indicadores y Reactivos/química , Indicadores y Reactivos/metabolismo , Indicadores y Reactivos/provisión & distribución , Técnicas de Diagnóstico Molecular , Plásmidos/química , Plásmidos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Biología Sintética/métodos , Transformación Bacteriana , Reino Unido/epidemiología
2.
Sci Rep ; 11(1): 9387, 2021 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-33931684

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5' untranslated region (5' UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.


Asunto(s)
/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /aislamiento & purificación , Animales , Tampones (Química) , Cricetinae , Humanos , Aplicaciones Móviles , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Factores de Tiempo
3.
Medicine (Baltimore) ; 100(20): e25916, 2021 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-34011059

RESUMEN

ABSTRACT: The outbreak and widely spread of coronavirus disease 2019 (COVID-19) has become a global public health concern. COVID-19 has caused an unprecedented and profound impact on the whole world, and the prevention and control of COVID-19 is a global public health challenge remains to be solved. The retrospective analysis of the large scale tests of SARS-CoV-2 RNA may indicate some important information of this pandemic. We selected 12400 SARS-CoV-2 tests detected in Wuhan in the first semester of 2020 and made a systematic analysis of them, in order to find some beneficial clue for the consistent prevention and control of COVID-19.SARS-CoV-2 RNA was detected in suspected COVID-19 patients with real-time fluorescence quantitative PCR (RT-qPCR). The patients' features including gender, age, type of specimen, source of patients, and the dynamic changes of the clinical symptoms were recorded and statistically analyzed. Quantitative and qualitive statistical analysis were carried out after laboratory detection.The positive rate of SARS-CoV-2 was 33.02% in 12,400 suspected patients' specimens in Wuhan at the first months of COVID-19 epidemics. SARS-CoV-2 RT-qPCR test of nasopharyngeal swabs might produce 4.79% (594/12400) presumptive results. The positive rate of SARS-CoV-2 RNA was significantly different between gender, age, type of specimen, source of patients, respectively (P < .05). The median window period from the occurrence of clinical symptom or close contact with COVID-19 patient to the first detection of positive PCR was 2 days (interquartile range, 1-4 days). The median interval time from the first SARS-CoV-2 positive to the turning negative was 14 days (interquartile range, 8-19.25 days).This study reveals the comprehensive characteristics of the SARS-CoV-2 RNA detection from multiple perspectives, and it provides important clues and may also supply useful suggestions for future work of the prevention and treatment of COVID-19.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/estadística & datos numéricos , COVID-19/diagnóstico , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/estadística & datos numéricos , SARS-CoV-2/genética , Adulto , Anciano , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19/métodos , China/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estudios Retrospectivos
4.
BMC Infect Dis ; 21(1): 463, 2021 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-34020607

RESUMEN

BACKGROUND: Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection ('challenge') model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis. METHODS: Combined sequential RNA + DNA extraction from throat swabs was compared to traditional separate RNA-only and DNA-only extractions. An emm-type specific qPCR was developed to detect the emm75 challenge strain. Results from the qPCR were compared to culture, using throat swab samples collected in a human challenge study. RESULTS: The qPCR was 100% specific for the emm75 challenge strain when tested against a panel of S. pyogenes emm-types and other respiratory pathogens. Combined RNA + DNA extraction had similar yield to traditional separate extractions. The combined extraction method and emm75 qPCR had 98.8% sensitivity compared to culture for throat swabs collected from challenge study participants. CONCLUSIONS: We have developed a reliable molecular method for measuring S. pyogenes bacterial load from throat swabs collected in a controlled human infection model of S. pyogenes pharyngitis. TRIAL REGISTRATION: NCT03361163 on 4th December 2017.


Asunto(s)
Antígenos Bacterianos/genética , Carga Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Faringitis/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Adulto , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Estudios de Seguimiento , Voluntarios Sanos , Humanos , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , Sensibilidad y Especificidad , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/aislamiento & purificación
5.
BMC Cancer ; 21(1): 439, 2021 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-33879115

RESUMEN

BACKGROUND: Routine clinical management of breast cancer (BC) currently depends on surrogate subtypes according to estrogen- (ER) and progesterone (PR) receptor, Ki-67, and HER2-status. However, there has been growing demand for reduced immunohistochemistry (IHC) turnaround times. The Xpert® Breast Cancer STRAT4* Assay (STRAT4)*, a standardized test for ESR1/PGR/MKi67/ERBB2 mRNA biomarker assessment, takes less than 2 hours. Here, we compared the concordance between the STRAT4 and IHC/SISH, thereby evaluating the effect of method choice on surrogate subtype assessment and adjuvant treatment decisions. METHODS: In total, 100 formalin-fixed paraffin-embedded core needle biopsy (CNB) samples and matching surgical specimens for 98 patients with primary invasive BC were evaluated using the STRAT4 assay. The concordance between STRAT4 and IHC was calculated for individual markers for the CNB and surgical specimens. In addition, we investigated whether changes in surrogate BC subtyping based on the STRAT4 results would change adjuvant treatment recommendations. RESULTS: The overall percent agreement (OPA) between STRAT4 and IHC/SISH ranged between 76 and 99% for the different biomarkers. Concordance for all four biomarkers in the surgical specimens and CNBs was only 66 and 57%, respectively. In total, 74% of surgical specimens were concordant for subtype, regardless of the method used. IHC- and STRAT4-based subtyping for the surgical specimen were shown to be discordant for 25/98 patients and 18/25 patients would theoretically have been recommended a different adjuvant treatment, primarily receiving more chemotherapy and trastuzumab. CONCLUSIONS: A comparison of data from IHC/in situ hybridization and STRAT4 demonstrated that subsequent changes in surrogate subtyping for the surgical specimen may theoretically result in more adjuvant treatment given, primarily with chemotherapy and trastuzumab.


Asunto(s)
Biomarcadores de Tumor , Biopsia con Aguja Gruesa , Neoplasias de la Mama/diagnóstico , Inmunohistoquímica , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Gruesa/métodos , Biopsia con Aguja Gruesa/normas , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/cirugía , Femenino , Humanos , Inmunohistoquímica/métodos , Inmunohistoquímica/normas , Mastectomía/métodos , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
6.
PLoS One ; 16(4): e0245423, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33852576

RESUMEN

BACKGROUND: In order to identify an inexpensive yet highly stable SARS-CoV-2 collection device as an alternative to foam swabs stored in transport media, both contrived ("surrogate") CoV-positive and patient-collected spun polyester swabs stored in dry tubes were evaluated for time- and temperature-stability using qPCR. METHODS: Surrogate specimens were prepared by combining multiple, residual SARS-CoV-2-positive clinical specimens and diluting to near-LOD levels in either porcine or human mucus ("matrix"), inoculating foam or polyester nasal swabs, and sealing in dry tubes. Swabs were then subjected to one of three temperature excursions: (1) 4°C for up to 72 hours; (2) 40°C for 12 hours, followed by 32°C for up to 60 hours; or (3) multiple freeze-thaw cycles (-20°C). The stability of extracted SARS-CoV-2 RNA for each condition was evaluated by qPCR. Separate usability studies for the dry polyester swab-based HealthPulse@home COVID-19 Specimen Collection Kit were later conducted in both adult and pediatric populations. RESULTS: Polyester swabs stored dry demonstrated equivalent performance to foam swabs for detection of low and moderate SARS-CoV-2 viral loads. Mimicking warm- and cold- climate shipment, surrogate specimens were stable following either 72 hours of a high-temperature excursion or two freeze-thaw cycles. In addition, usability studies comprised of self-collected patient specimens yielded sufficient material for molecular testing, as demonstrated by RNase P detection. CONCLUSIONS: Polyester nasal swabs stored in dry collection tubes offer a robust and inexpensive self-collection method for SARS-CoV-2 viral load testing, as viral RNA remains stable under conditions required for home collection and shipment to the laboratory.


Asunto(s)
/diagnóstico , /aislamiento & purificación , Manejo de Especímenes/métodos , Animales , Técnicas de Laboratorio Clínico/métodos , Pruebas Diagnósticas de Rutina/métodos , Humanos , Técnicas de Diagnóstico Molecular , Nasofaringe/virología , Poliésteres , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Porcinos
7.
PLoS One ; 16(4): e0243333, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33852580

RESUMEN

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.


Asunto(s)
/diagnóstico , Técnicas Analíticas Microfluídicas/métodos , Manejo de Especímenes/métodos , Adulto , /métodos , Cartilla de ADN , Pruebas Diagnósticas de Rutina/métodos , Femenino , Humanos , Masculino , MicroARNs/genética , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad
8.
Viruses ; 13(4)2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33810324

RESUMEN

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.


Asunto(s)
/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /genética , Adulto , Anciano , Anciano de 80 o más Años , /normas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , /fisiología , Adulto Joven
9.
Acta Med Indones ; 53(1): 13-17, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33818402

RESUMEN

BACKGROUND: real-time RT-PCR was recommended by WHO for COVID-19 diagnosis. The cycle threshold (Ct) values were expected to have an association with clinical manifestation. However, the diagnostic modalities such as quantitative molecular detection and virus isolation were not yet available for the routine test. This study has been conducted to analyze the relationship between the Ct values of qualitative rRT-PCR and the clinical manifestation and to describe the factors determining the result. METHODS: from March to April 2020, specimens were sent to our laboratory from different healthcare centers in Jakarta. The patient's characteristic and clinical manifestation were extracted from the specimen's epidemiology forms. The specimens extracted and tested using rRT-PCR, and the Ct value were collected. The data were analyzed using the appropriate statistic test. RESULTS: from 339 positive results, the mild to moderate case was 176 (52%) and the severe cases was 163 (48%). Female was dominant in the mild to moderate cases (58%), while the male was prevalent in the severe cases (60%). The median age for mild to moderate case was 35 years old and severe cases was 49 years old. Statistical analysis found relationship between both group with gender (p = 0.001) and age (p < 0.001), but not with the Ct value. CONCLUSION: many variables in specimen sampling and processing could affect the Ct value result. In addition, the disease's severity was depended with the host immune response, regardless the number of virus. There was suggested no significant difference between the Ct values of mild-moderate and severe COVID-19, and thus should not be loosely interpreted.


Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa , Evaluación de Síntomas , Adulto , Factores de Edad , /epidemiología , /métodos , /estadística & datos numéricos , Correlación de Datos , Femenino , Humanos , Indonesia/epidemiología , Masculino , Persona de Mediana Edad , Variaciones Dependientes del Observador , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/estadística & datos numéricos , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Factores Sexuales , Evaluación de Síntomas/métodos , Evaluación de Síntomas/estadística & datos numéricos , Carga Viral
10.
Viruses ; 13(5)2021 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-33922195

RESUMEN

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.


Asunto(s)
Prueba de COVID-19/métodos , COVID-19/diagnóstico , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , COVID-19/virología , Femenino , Genes Virales , Genoma Viral , Humanos , Masculino , ARN Viral/análisis , ARN Viral/genética , SARS-CoV-2/aislamiento & purificación
11.
Genes (Basel) ; 12(4)2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33924636

RESUMEN

For 1 year now, the world is undergoing a coronavirus disease-2019 (COVID-19) pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method for COVID-19 diagnosis is the detection of viral RNA by RT-qPCR with a specific set of primers and probe. It is important to frequently evaluate the performance of these tests and this can be done first by an in silico approach. Previously, we reported some mismatches between the oligonucleotides of publicly available RT-qPCR assays and SARS-CoV-2 genomes collected from GISAID and NCBI, potentially impacting proper detection of the virus. In the present study, 11 primers and probe sets investigated during the first study were evaluated again with 84,305 new SARS-CoV-2 unique genomes collected between June 2020 and January 2021. The lower inclusivity of the China CDC assay targeting the gene N has continued to decrease with new mismatches detected, whereas the other evaluated assays kept their inclusivity above 99%. Additionally, some mutations specific to new SARS-CoV-2 variants of concern were found to be located in oligonucleotide annealing sites. This might impact the strategy to be considered for future SARS-CoV-2 testing. Given the potential threat of the new variants, it is crucial to assess if they can still be correctly targeted by the primers and probes of the RT-qPCR assays. Our study highlights that considering the evolution of the virus and the emergence of new variants, an in silico (re-)evaluation should be performed on a regular basis. Ideally, this should be done for all the RT-qPCR assays employed for SARS-CoV-2 detection, including also commercial tests, although the primer and probe sequences used in these kits are rarely disclosed, which impedes independent performance evaluation.


Asunto(s)
/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /genética , Simulación por Computador , Genoma Viral , Humanos , Mutación , Sondas ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos
12.
Pathol Res Pract ; 221: 153443, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33930607

RESUMEN

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the control of virus spread has remained challenging given the pitfalls of the current diagnostic tests. Nevertheless, RNA amplification techniques have been the gold standard among other diagnostic methods for monitoring clinical samples for the presence of the virus. In the current paper, we review the shortcomings and strengths of RT-PCR (real-time polymerase chain reaction) techniques for diagnosis of coronavirus disease (COVID)-19. We address the repercussions of false-negative and false-positive rates encountered in the test, summarize approaches to improve the overall sensitivity of this method. We discuss the barriers to the widespread use of the RT-PCR test, and some technical advances, such as RT-LAMP (reverse-transcriptase-loop mediated isothermal amplification). We also address how other molecular techniques, such as immunodiagnostic tests can be used to avoid incorrect interpretation of RT-PCR tests.


Asunto(s)
/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /aislamiento & purificación , Humanos
13.
Genes (Basel) ; 12(5)2021 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-33924826

RESUMEN

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization-World Health Organization-International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Argentina , Calibración , Humanos , Límite de Detección , /genética
14.
Am J Clin Pathol ; 155(6): 815-822, 2021 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-33822853

RESUMEN

OBJECTIVES: The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR-in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. METHODS: We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer's specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription-polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. RESULTS: The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. CONCLUSIONS: The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.


Asunto(s)
/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Humanos , Límite de Detección , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Carga Viral/métodos
15.
Int J Infect Dis ; 106: 421-428, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33794378

RESUMEN

BACKGROUND: Pertussis is an acute respiratory tract disease caused by Bordetella pertussis. In 2014, 24.1 million pertussis cases, resulting in 160,700 deaths, were estimated to have occurred worldwide. This study aimed to determine the epidemiology of pertussis among patients with clinically compatible illness who visited selected hospitals in the Amhara Regional State of Ethiopia. METHODS: A cross-sectional study design was used to review pertussis patients with clinically compatible illness. Nasopharyngeal swabs were collected from 515 patients from July 2018 through February 2019. DNA was extracted from all nasopharyngeal swabs and samples were analyzed using real-time (RT-) PCR. Crude and adjusted odds ratios with corresponding 95% confidence intervals were estimated using bivariable and multivariable logistic regression analysis, respectively. RESULTS: The overall prevalence of Bordetella species among the study participants was 156 of 515 (30.3%) [95% CI = 26.4-34.6] as determined by Bordetella RT-PCR, including: 65 (41.7%) B. pertussis, 89 (57.1%) indeterminate B. pertussis, one (0.6%) Bordetella holmesii and one (0.6%) Bordetella parapertussis. CONCLUSIONS: This study found that pertussis is potentially endemic and a common health problem among patients visiting health institutions in the Amhara Regional State of Ethiopia. More data regarding pertussis in Ethiopia could inform development of effective prevention strategies.


Asunto(s)
Tos Ferina/epidemiología , Adulto , Bordetella pertussis , Estudios Transversales , Pruebas Diagnósticas de Rutina , Etiopía/epidemiología , Humanos , Masculino , Prevalencia , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos
16.
PLoS Negl Trop Dis ; 15(3): e0009227, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33647020

RESUMEN

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/µl viral RNA for the UpE assay and 1.2 copies/µl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/virología , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Técnicas de Diagnóstico Molecular/métodos , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Humanos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcripción Reversa , Arabia Saudita/epidemiología , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/genética
17.
Methods Mol Biol ; 2292: 17-22, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33651348

RESUMEN

Urine cell-free DNA is an important source of diagnostic markers for different diseases, especially for cancer. It could be important to achieve the urine cell-free DNA integrity to establish its provenience from cancer cells or dead inflammatory cells for necrosis in urine or from normal cells with the purpose to use it as an early diagnostic tool for urological cancers or other diseases. Here we describe a simple, noninvasive approach from urine collection to DNA integrity analysis using real-time PCR.


Asunto(s)
Ácidos Nucleicos Libres de Células/orina , Neoplasias/orina , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/orina , Ácidos Nucleicos Libres de Células/genética , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Humanos , Neoplasias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Toma de Muestras de Orina/métodos
18.
Methods Mol Biol ; 2292: 23-33, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33651349

RESUMEN

Urinary cell-free DNA offers an important noninvasive source of material for genomic testing also for nonurological tumors. Its clinical utility in monitoring tumor evolution and treatment failure is promising. Here we describe a method to detect cancer mutations into urine from patients affected by colorectal cancer.


Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN/métodos , Proteínas Proto-Oncogénicas p21(ras)/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/orina , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Ácidos Nucleicos Libres de Células/orina , Neoplasias Colorrectales/orina , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos
19.
Methods Mol Biol ; 2292: 49-56, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33651351

RESUMEN

Urine cell-free DNA has been shown as an informative noninvasive source of biomarkers for a number of diseases, especially for urological cancers. Starting from the hypothesis that the gain of c-Myc gene is a frequent aberration in several cancer types, including prostate cancer, we analyzed c-Myc copy number variation in urine, studying a little case series of prostate cancer patients, to test its feasibility. Here we report a general protocol that may be considered to analyze gene copy number variation in the urine cell-free fraction.


Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Variaciones en el Número de Copia de ADN , Genes myc , Neoplasias de la Próstata/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/orina , Ácidos Nucleicos Libres de Células/orina , Humanos , Masculino , Neoplasias de la Próstata/orina
20.
Methods Mol Biol ; 2292: 133-141, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33651358

RESUMEN

Bladder cancer with an incidence of 15 cases per 100,000 persons in the global population is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers is still an urgency. Among the biomarkers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Previous studies have shown that the quantitative telomerase repeat amplification protocol (TRAP) assay performed in voided urine is an important noninvasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low-grade tumors. Telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost-benefit terms when used in selected symptomatic patients or professionally high-risk subgroups. Here we report the real-time PCR protocol to detect telomerase activity in urine sediment for bladder cancer.


Asunto(s)
Telomerasa/orina , Neoplasias de la Vejiga Urinaria/orina , Biomarcadores de Tumor/orina , Pruebas de Enzimas/métodos , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Toma de Muestras de Orina/métodos
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