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1.
Biomed Res Int ; 2020: 7610678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029522

RESUMO

Background: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. Methods: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. Results: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. Conclusions: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Pneumonia Viral/diagnóstico , Sondas RNA/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
2.
Artigo em Inglês | MEDLINE | ID: mdl-33053146

RESUMO

Meningoencephalitis is a syndrome of multiple etiologies associated with important morbidity and mortality. It may be caused by various infectious agents (viruses, bacteria, parasites and fungi). Establishing the etiology of meningoencephalitis is crucial for early and specific treatment. Molecular assays such as the multiplex polymerase chain reaction (PCR) offer an alternative in diagnosing central nervous system infections. This study aimed to describe the performance of an automated multiplex molecular test from patients with suspected meningitis and meningoencephalitis in a tertiary referral complex in Medellin, Colombia. Thus, a prospective study was performed in 638 cerebrospinal fluid samples from January 2017 to July 2019. Molecular detections were carried out by means of the FilmArray® Meningitis/Encephalitis (M/E) Panel from bioMérieux, France, and by conventional tests. Univariate analyses for microbiological and demographic characteristics were performed. Accuracy of the bacterial/fungal PCR assay compared to cultures was also performed. Among patients, 57.7% were male, the median age was 24 (IQR: 6 - 47) years old. The overall positivity was 15.2% (97 detections) and viruses were detected in 45.5% of the samples, bacteria in 43.5% and fungi in 10.8%. The most frequent etiological agents were: Streptococcus pneumoniae (16%), Cryptococcus neoformans/gatti (11.3%) and Herpes simplex virus (10.3%). Four double detections were found. Almost half of positive detections were in patients under 15 years old. This molecular approach is reliable and easily implantable into a laboratory routine, increasing the capacity of detection of bacterial and viral causative agents of meningitis, possibly playing a relevant role in the clinical context.


Assuntos
Meningite/epidemiologia , Meningoencefalite/epidemiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Adolescente , Adulto , Criança , Colômbia/epidemiologia , Feminino , Humanos , Masculino , Meningite/etiologia , Meningoencefalite/etiologia , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
3.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946527

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
4.
Viruses ; 12(8)2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824272

RESUMO

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , DNA Complementar/genética , Genoma Viral , Humanos , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Sequenciamento Completo do Genoma/métodos
5.
J Appl Lab Med ; 5(5): 897-907, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32674131

RESUMO

BACKGROUND: Upper respiratory tract infections are common, and the ability to accurately and rapidly diagnose the causative pathogen has important implications for patient management. METHODS: We evaluated the test-ordering practices for 2 commonly utilized nucleic acid amplification tests (NAATs) for the detection of respiratory pathogens: the Xpert Flu Assay for influenza A/B (Flu assay) and the Biofire FilmArray respiratory panel assay (RP assay), which detects 20 different targets. Our study examined repeat testing; that is, testing within 7 days from an initial test. RESULTS: Our study found that repeat testing is common for each of the individual assays: 3.0% of all Flu assays and 10.0% of all RP assays were repeat testing. Of repeat testing, 8/293 (2.7%) of repeat Flu assays and 75/1257 (6.0%) of RP assays resulted diagnostic gains, i.e., new detections. However, for the RP assay, these new detections were not always clinically actionable. The most frequently discrepant organisms were rhinovirus/enterovirus (28/102, 27.5%), followed by respiratory syncytial virus (12/102, 11.8%) and coronavirus OC43 (11/102, 10.8%). Furthermore, there were 3,336 instances in which a patient was tested using both a Flu assay and RP assay, of which only 44 (1.3%) had discrepant influenza results. CONCLUSIONS: Our findings suggest opportunities exist to better guide ordering practices for respiratory pathogen testing, including limiting repeat testing, with the goal of optimization of clinical yield, and diagnostic stewardship.


Assuntos
Vírus da Influenza A , Vírus da Influenza B , Influenza Humana , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Vírus de RNA , Infecções Respiratórias , Diagnóstico Diferencial , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Utilização de Procedimentos e Técnicas , Infecções por Vírus de RNA/diagnóstico , Infecções por Vírus de RNA/virologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Vírus/classificação , Vírus/isolamento & purificação
6.
BMC Infect Dis ; 20(1): 550, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727378

RESUMO

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N8/genética , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Neuraminidase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aves/virologia , Primers do DNA/genética , Feminino , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Biosens Bioelectron ; 166: 112437, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32692666

RESUMO

The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/estatística & dados numéricos , China/epidemiologia , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Desenho de Equipamento , Estudos de Viabilidade , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Nanopartículas , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade
8.
J Vis Exp ; (160)2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32658205

RESUMO

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.


Assuntos
Metilação de DNA/fisiologia , Testes Hematológicos/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Polímeros/química , Humanos , Leucócitos Mononucleares/química
9.
Genome Med ; 12(1): 57, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32605661

RESUMO

BACKGROUND: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. METHODS: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. RESULTS: We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. CONCLUSIONS: This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.


Assuntos
Betacoronavirus/genética , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento Completo do Genoma/métodos , Infecções por Coronavirus , Variação Genética/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Pneumonia Viral , RNA Viral/genética
10.
PLoS Negl Trop Dis ; 14(6): e0008392, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542036

RESUMO

The canine hookworms Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and mortality in dogs but are also neglected tropical zoonoses. Each hookworm species differs considerably in its geographical distribution, life cycle, biology, pathogenic impacts on both canine and human hosts, zoonotic potential, and response to treatment with anthelminthics. Here we describe the development and validation of two Taq-Man based multiplex PCR assays capable of detecting and differentiating all four canine hookworm species in faeces of naturally infected dogs. The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each hookworm species. The sensitivity of the assays and ability to detect mixed species infections were compared to a conventional PCR-Restriction Fragment Length Polymorphism based-approach when applied to laboratory and field samples from endemic areas. The qPCRs detected at least one species of hookworms in 82.4% of PCR-RFLP-negative but microscopy-positive samples. The qPCRs detected an additional 68% mixed infections with different species of canine hookworms, and additional single species infection with A. caninum (47%), U. stenocephala (33%) and A. ceylanicum (0.02%) that were missed by PCR-RFLP. These multiplex qPCR assays will assist field based epidemiological surveillance studies towards an accurate and sensitive monitoring of canine hookworm infections in dogs, to inform their species-specific zoonotic risks to populations living in endemic areas, globally.


Assuntos
Ancylostomatoidea/genética , Ancylostomatoidea/isolamento & purificação , Doenças do Cão/diagnóstico , Infecções por Uncinaria/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária , Ancylostoma/genética , Ancylostoma/isolamento & purificação , Ancylostomatoidea/classificação , Ancilostomíase/diagnóstico , Ancilostomíase/epidemiologia , Ancilostomíase/fisiopatologia , Animais , DNA de Helmintos/análise , DNA de Helmintos/genética , Doenças do Cão/epidemiologia , Doenças do Cão/fisiopatologia , Cães , Fezes/parasitologia , Infecções por Uncinaria/fisiopatologia , Humanos , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade , Zoonoses/diagnóstico , Zoonoses/epidemiologia , Zoonoses/fisiopatologia
11.
Exp Mol Med ; 52(6): 963-977, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32546849

RESUMO

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Primers do DNA , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Infecções por Coronavirus/diagnóstico , Células HEK293 , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Faringe/virologia , Pneumonia Viral/diagnóstico , RNA Replicase/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vírus da SARS/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
12.
Euro Surveill ; 25(24)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32583765

RESUMO

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Equipamentos para Diagnóstico , Pneumonia Viral/diagnóstico , Técnicas de Laboratório Clínico/instrumentação , Fezes/virologia , Alemanha , Humanos , Laboratórios , Reação em Cadeia da Polimerase Multiplex/instrumentação , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
13.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-361278

RESUMO

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Reação em Cadeia da Polimerase Multiplex , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Primers do DNA , Sondas de DNA , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Carga Viral
14.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32448084

RESUMO

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Reação em Cadeia da Polimerase Multiplex , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Primers do DNA , Sondas de DNA , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Carga Viral
15.
Exp Parasitol ; 215: 107918, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464220

RESUMO

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.


Assuntos
Culex/parasitologia , Filariose Linfática/transmissão , Mosquitos Vetores/parasitologia , Reação em Cadeia da Polimerase Multiplex/métodos , Wuchereria bancrofti/fisiologia , Actinas/genética , Animais , Sequência de Bases , Eletroforese em Gel de Ágar , Filariose Linfática/sangue , Filariose Linfática/tratamento farmacológico , Filariose Linfática/prevenção & controle , Feminino , Humanos , Doenças Negligenciadas/parasitologia , Sensibilidade e Especificidade , Wuchereria bancrofti/genética
16.
J Med Microbiol ; 69(5): 712-720, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32368996

RESUMO

Introduction. Given the limited number of candidaemia studies in Iran, the profile of yeast species causing bloodstream infections (BSIs), especially in adults, remains limited. Although biochemical assays are widely used in developing countries, they produce erroneous results, especially for rare yeast species.Aim. We aimed to assess the profile of yeast species causing BSIs and to compare the accuracy of the Vitek 2 system and 21-plex PCR.Methodology. Yeast blood isolates were retrospectively collected from patients recruited from two tertiary care training hospitals in Tehran from 2015 to 2017. Relevant clinical data were mined. Identification was performed by automated Vitek 2, 21-plex PCR and sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2).Results. In total, 137 yeast isolates were recovered from 107 patients. The overall all-cause 30-day mortality rate was 47.7 %. Fluconazole was the most widely used systemic antifungal. Candida albicans (58/137, 42.3 %), Candida glabrata (30/137, 21.9 %), Candida parapsilosis sensu stricto (23/137, 16.8 %), Candida tropicalis (10/137, 7.3 %) and Pichia kudriavzevii (Candida krusei) (4/137, 2.9 %) constituted almost 90 % of the isolates and 10 % of the species detected were rare yeast species (12/137; 8.7 %). The 21-plex PCR method correctly identified 97.1 % of the isolates, a higher percentage than the Vitek 2 showed (87.6 %).Conclusion. C. albicans was the main cause of yeast-derived fungaemia in this study. Future prospective studies are warranted to closely monitor the epidemiological landscape of yeast species causing BSIs in Iran. The superiority of 21-plex PCR over automated Vitek 2 indicates its potential clinical utility as an alternative identification tool use in developing countries.


Assuntos
Fungemia/diagnóstico , Fungemia/epidemiologia , Fungemia/microbiologia , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência de DNA , Leveduras/classificação , Leveduras/genética , Idoso , Idoso de 80 Anos ou mais , DNA Intergênico , Feminino , Fungemia/história , História do Século XXI , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
17.
Nat Commun ; 11(1): 2607, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451375

RESUMO

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.


Assuntos
Citocinas/sangue , DNA Bacteriano/sangue , Choque Séptico/imunologia , Choque Séptico/microbiologia , Asma/imunologia , Asma/microbiologia , Carga Bacteriana , Biomarcadores/análise , Biomarcadores/sangue , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Estudos de Casos e Controles , Citocinas/análise , DNA Bacteriano/genética , Árvores de Decisões , Genes Bacterianos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interleucina-6/análise , Interleucina-6/sangue , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Prognóstico , Sensibilidade e Especificidade , Choque Séptico/mortalidade , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangue , Resistência beta-Lactâmica/genética
18.
J Clin Virol ; 128: 104448, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32460173

RESUMO

BACKGROUND: In the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays. AIMS: To evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2. METHODS: We reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory. RESULTS: Initial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results. CONCLUSION: The AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Pandemias , Pneumonia Viral/diagnóstico , Adulto , Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
Emerg Infect Dis ; 26(7): 1633-1635, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32294051

RESUMO

Most reverse transcription PCR protocols for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 2-3 targets for detection. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with singleplex assays. This protocol could streamline detection and decrease reagent use during current high SARS-CoV-2 testing demands.


Assuntos
Betacoronavirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Humanos , Nasofaringe/virologia
20.
PLoS One ; 15(4): e0232050, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324777

RESUMO

BACKGROUND: Next-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA genes in one PCR reaction. Here, we report the performance of this unique workflow evaluated using 218 genetically diverse samples. METHODS: Five whole genes (HLA-A/B/C/DQA1/DPA1) and six near-whole genes (HLA-DRB1/DRB345/DQB1/DPB1; excluding exon 1 and part of intron 1) were amplified in a multiplexed, long-range PCR. Manual library preparation was performed per manufacturer's protocol, followed by template preparation and chip loading on the Ion Chef, and sequencing on the Ion S5 sequencer. Pre-specified rules for quality control and repeat testing were followed; technologists were blinded to the reference results. The concordance between AllType and reference results was determined at 2-field resolution. We also describe the ranges of input DNA and library concentrations, read number per sample and per locus, and key health metrics in relation to typing results. RESULTS: The concordance rates were 98.6%, 99.8% and 99.9% at the sample (n = 218), genotype (n = 1688), and allele (n = 3376) levels, respectively. Three genotypes were discordant, all of which shared the same G group typing results with the reference. Most ambiguous genotypes (116 out of 144, 80.6%) were due to the lack of exon 1 and intron 1 coverage for HLA-DRB1/DRB345/DQB1/DPB1 genes. A broad range of input DNA concentrations and library concentrations were tolerated. Per sample read numbers were adequate for accurate genotyping. Per locus read numbers showed some inter-lot variations, and a trend toward improved inter-locus balance was observed with later lots of reagents. CONCLUSION: The AllType assay on the Ion Chef/Ion S5 platform offers a robust and efficient workflow for clinical HLA typing at the 2-field resolution. The multiplex PCR strategy simplifies the laboratory procedure without compromising the typing accuracy.


Assuntos
Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Humanos , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase Multiplex/métodos , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Fluxo de Trabalho
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