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A method of processing nasopharyngeal swabs to enable multiple testing.

ABSTRACT: OBJECTIVE:To develop a method to perform multiple tests on a single nasopharyngeal (NP) swab. METHODS:We collected a NP swab on children aged 2-12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA-sequencing, which we accomplished by cutting the tip of the swab. RESULTS:Of the 174 children enrolled, 126 (72.4%) had a positive bacterial culture and 121 (69.5%) tested positive for a virus. Cytokine measurement, as judged by adequate levels of a housekeeping enzyme (glyceraldehyde 3-phosphate dehydrogenase), appeared successful. From the samples used for 16S ribosomal sequencing we recovered, on average, 16,000 sequences per sample, accounting for a total of 2646 operational taxonomic units across all samples sequenced. Samples used for RNA-sequencing had a mean RNA integrity number of 6.0. Cutting the tip of the swab did not affect the recovery yield for viruses or bacteria, nor did it affect species richness in microbiome analysis. CONCLUSION:We describe a minimally invasive sample collection protocol that allows for multiple diagnostic and research investigations in young children.

SUBMITTER: Lopez SMC

PROVIDER: S-EPMC6851467 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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A method of processing nasopharyngeal swabs to enable multiple testing.

Lopez Santiago M C SMC Martin Judith M JM Johnson Monika M Kurs-Lasky Marcia M Horne William T WT Marshall Christopher W CW Cooper Vaughn S VS Williams John V JV Shaikh Nader N

Pediatric research 20190709 5

<h4>Objective</h4>To develop a method to perform multiple tests on a single nasopharyngeal (NP) swab.<h4>Methods</h4>We collected a NP swab on children aged 2-12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA-sequencing, which we accomplished by cutting the tip of the swab.<h4>Results</h4>Of the 174 children enrolled, 1 ...[more]

PMID: 31288247

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Project description:Numerous reverse transcription polymerase chain reaction (RT-PCR) tests have emerged over the past year as the gold standard for detecting millions of cases of SARS-CoV-2 reported daily worldwide. However, problems with critical shortages of key reagents such as PCR primers and RNA extraction kits and unpredictable test reliability related to high viral replication cycles have triggered the need for alternative methodologies to PCR to detect specific COVID-19 proteins. Several authors have developed methods based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to confirm the potential of the technique to detect two major proteins, the spike and the nucleoprotein, of COVID-19. In the present work, an S-Trap mini spin column digestion protocol was used for sample preparation prodromal to LC-MS/MS analysis in multiple reactions monitoring ion mode (MRM) to obtain a comprehensive method capable of detecting different viral proteins. The developed method was applied to n. 81 oro/nasopharyngeal swabs submitted in parallel to quantitative reverse transcription PCR (RT-qPCR) assays to detect RdRP, the S and N genes specific for COVID-19, and the E gene for all Sarbecoviruses, including SARS-CoV-2 (with cycle negativity threshold set to 40). A total of 23 peptides representative of the six specific viral proteins were detected in the monitoring of 128 transitions found to have good ionic currents extracted in clinical samples that reacted differently to the PCR assay. The best instrumental response came from the FLPFQFGR sequence of spike [558-566] peptide used to test the analytical performance of the method that has good sensitivity with a low false-negative rate. Transition monitoring using a targeted MS approach has the great potential to detect the fragmentation reactions of any peptide molecularly defined by a specific amino acid sequence, offering the extensibility of the approach to any viral sequence including derived variants and thus providing insights into the development of new types of clinical diagnostics.

Project description:BackgroundThe impact of COVID-19 on the world is still ongoing, and it is currently under regular management. Although most infected people have flu-like symptoms and can cure themselves, coexisting pathogens in COVID-19 patients should not be taken lightly. The present study sought to investigate the coexisting pathogens in SARS-CoV-2 infected patients and identify the variety and abundance of dangerous microbes to guide treatment strategies with a better understanding of the untested factors.MethodsWe extracted total DNA and RNA in COVID-19 patient specimens from nasopharyngeal swabs to construct a metagenomic library and utilize Next Generation Sequencing (NGS) to discover chief bacteria, fungi, and viruses in the body of patients. High-throughput sequencing data from Illumina Hiseq 4000 were analyzed using Krona taxonomic methodology for species diversity.ResultsWe studied 56 samples to detect SARS-CoV-2 and other pathogens and analyzed the species diversity and community composition of these samples after sequencing. Our results showed some threatening pathogens such as Mycoplasma pneumoniae, Klebsiella pneumoniae, Streptococcus pneumoniae, and some previously reported pathogens. SARS-CoV-2 combined with bacterial infection is more common. The results of heat map analysis showed that the abundance of bacteria was mostly more than 1000 and that of viruses was generally less than 500. The pathogens most likely to cause SARS-CoV-2 coinfection or superinfection include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Klebsiella pneumoniae, and Human gammaherpesvirus 4.ConclusionsThe current coinfection and superinfection status is not optimistic. Bacteria are the major threat group that increases the risk of complications and death in COVID-19 patients and attention should be paid to the use and control of antibiotics. Our study investigated the main types of respiratory pathogens prone to coexisting or superinfection in COVID-19 patients, which is valuable for identifying and treating SARS-CoV-2.

Project description:BackgroundNasopharyngeal swabs are the primary sampling method used for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but they require a trained health care professional and extensive personal protective equipment.PurposeTo determine the difference in sensitivity for SARS-CoV-2 detection between nasopharyngeal swabs and saliva and estimate the incremental cost per additional SARS-CoV-2 infection detected with nasopharyngeal swabs.Data sourcesEmbase, Medline, medRxiv, and bioRxiv were searched from 1 January to 1 November 2020. Cost inputs were from nationally representative sources in Canada and were converted to 2020 U.S. dollars.Study selectionStudies including at least 5 paired nasopharyngeal swab and saliva samples and reporting diagnostic accuracy for SARS-CoV-2 detection.Data extractionData were independently extracted using standardized forms, and study quality was assessed using QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies 2).Data synthesisThirty-seven studies with 7332 paired samples were included. Against a reference standard of a positive result on either sample, the sensitivity of saliva was 3.4 percentage points lower (95% CI, 9.9 percentage points lower to 3.1 percentage points higher) than that of nasopharyngeal swabs. Among persons with previously confirmed SARS-CoV-2 infection, saliva's sensitivity was 1.5 percentage points higher (CI, 7.3 percentage points lower to 10.3 percentage points higher) than that of nasopharyngeal swabs. Among persons without a previous SARS-CoV-2 diagnosis, saliva was 7.9 percentage points less (CI, 14.7 percentage points less to 0.8 percentage point more) sensitive. In this subgroup, if testing 100 000 persons with a SARS-CoV-2 prevalence of 1%, nasopharyngeal swabs would detect 79 more (95% uncertainty interval, 5 fewer to 166 more) persons with SARS-CoV-2 than saliva, but with an incremental cost per additional infection detected of $8093.LimitationThe reference standard was imperfect, and saliva collection procedures varied.ConclusionSaliva sampling seems to be a similarly sensitive and less costly alternative that could replace nasopharyngeal swabs for collection of clinical samples for SARS-CoV-2 testing.Primary funding sourceMcGill Interdisciplinary Initiative in Infection and Immunity. (PROSPERO: CRD42020203415).

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A method of processing nasopharyngeal swabs to enable multiple testing.

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A method of processing nasopharyngeal swabs to enable multiple testing.

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