Myrsini Kaforou (United Kingdom)

Author Of 4 Presentations

A NOVEL AND POWER-FREE SAMPLE PREPARATION METHOD TO ALLOW RAPID DETECTION OF SARS-COV-2 RNA FROM NASOPHARYNGEAL SAMPLES

Date
Fri, 13.05.2022
Session Time
10:00 - 11:30
Session Type
Oral Presentations Session
Room
MC 2 HALL
Lecture Time
10:12 - 10:22

Abstract

Backgrounds:

The ongoing COVID-19 pandemic has led to over five million deaths worldwide highlighting an unprecedented need for rapid diagnostic screening. The gold standard for COVID-19 diagnosis is the collection of a nasopharyngeal swab subsequently processed with an RNA extraction kit requiring electricity and expensive laboratory equipment. Therefore, the diagnosis of COVID-19 in low- and middle-income countries (LMIC) is rarely achievable at a point-of-care (POC) and instead relegated to remote centralized laboratories. To address this need, our team has developed an innovative, rapid and easy to use sample preparation method for RNA extraction allowing for true POC application.

Methods

The SmartLidTM extraction method utilizes a custom 3D printed magnetic lid, designed to work with standard Eppendorf tubes, to transfer magnetic nanoparticles and attached RNA through three sample preparation steps. This is in contrast to all other manual extraction methods, which require expensive micropipettes, lab training and electrical power. The whole extraction process is performed within five minutes providing pure RNA adequate for downstream applications.

Results:

A total of 410 nasopharyngeal swabs has been tested (including 150 COVID-positive subjects). All clinical isolates were extracted by the SmartLid and the gold standard QIAmp Viral RNA methods and tested by the CDC RT-qPCR assays. The SmartLid method achieved 93.9% sensitivity and 99.5% specificity compared to the QIAmp Viral RNA showing equivalent performance.

Conclusions/Learning Points:

The extraction method presented can compete favourably with conventional laboratory-based extraction techniques which require expensive equipment and electricity, and which incur delays of over 45 minutes. The method has received overwhelmingly positive feedback from collaborators, who have tested it in CAT3 laboratory environments, and from visiting clinicians with experience in LMIC diagnostics. Thus, there is a clear interest for implementation in a domestic clinical/laboratory setting (NHS),and LMIC remote point-of-care setting.

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Precision Medicine in the Management of Fever: The Role of Host Biomarkers

Date
Wed, 11.05.2022
Session Time
15:40 - 17:15
Session Type
Parallel Symposium
Room
DIMITRIS MITROPOULOS HALL
Lecture Time
16:07 - 16:32

VALIDATION OF TRANSCRIPTOMIC SIGNATURES FOR FEBRILE CHILDREN USING NANOSTRING TECHNOLOGY AND EXPLORATION OF MULTI-CLASS PREDICTION MODELS

Date
Wed, 11.05.2022
Session Time
15:40 - 17:15
Session Type
Parallel Symposium
Room
DIMITRIS MITROPOULOS HALL
Lecture Time
16:53 - 17:01

Abstract

Backgrounds:

Many host transcript signatures for paediatric inflammatory and infectious diseases are in development, but require validation in independent cohorts; their translation to clinically useful test platforms lags behind discovery. We used NanoString technology to efficiently validate multiple signatures in parallel and explore the potential for more sophisticated multi-class classification models.

Methods

We validated five transcriptomic diagnostic signatures using prospectively recruited patients from multiple paediatric cohorts. Final phenotypes were assigned using pre-agreed definitions after review of clinical and laboratory data. We quantified 69 transcripts on a custom NanoString nCounter cartridge, normalising expression values using reference genes. Signature performance was assessed using Area Under ROC Curve (AUC) statistics. We explored two approaches to multiclassification diagnostics to develop proof-of-concept methods: a mixed test combining four independent one-vs-all models, and a multinomial model.

Results:

Our cohort of 92 paediatric patients included 23 definite bacterial and 20 definite viral infections, 15 Kawasaki disease, 18 with tuberculosis and 16 healthy controls. The signatures achieved AUCs above 0.82 (Table 1), with confidence intervals overlapping those of the respective discovery studies. However, performance declined in all signatures when tasked with differentiating additional groups. For example, the single-transcript BATF2 had AUC of 0.910 differentiating TB from healthy individuals, reducing to 0.745 when differentiating TB from other febrile diseases. In comparison, the multinomial approach identified a 24-transcript model that correctly classified all 76 non-control patients (0% in-sample error), outperforming the mixed-model (19 transcripts, 19.8% in-sample error).

table1.jpg

Conclusions/Learning Points:

The cross-platform, out-of-sample findings validated 5 signatures, but discriminatory power was reduced in patients drawn from outside their remit. An exploratory 24-transcript model had improved accuracy across all diagnostic groups, demonstrating in principle the utility for one-step multi-class diagnosis in patients with broad diagnostic uncertainty.

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THE IDENTIFICATION AND SUBSEQUENT CROSS-PLATFORM VALIDATION OF A HOST GENE EXPRESSION SIGNATURE FOR DIFFERENTIATING BETWEEN MIS-C AND OTHER INFECTIOUS AND INFLAMMATORY DISEASES

Date
Wed, 11.05.2022
Session Time
13:40 - 15:10
Session Type
Joint Symposium
Room
BANQUETING HALL
Lecture Time
14:58 - 15:06

Abstract

Backgrounds:

Multisystem Inflammatory Syndrome in Children (MIS-C) occurs several weeks after SARS-CoV-2 infection with symptoms including fever, shock and multiorgan failure. Clinical features of MIS-C overlap with Kawasaki Disease (KD), bacterial, and viral infections, making accurate diagnosis challenging. Host genes, measurable through whole blood transcriptomics, are an alternative tool for diagnosing infectious and inflammatory diseases.

Methods

Patients with MIS-C, KD, bacterial, and viral infections were recruited to the EU-funded PERFORM and DIAMONDS studies and the NIH-funded PREVAIL study. Patients were phenotyped using a standardised algorithm. Genome wide RNA sequencing of whole blood was undertaken, and feature selection was performed to identify a diagnostic signature for distinguishing between MIS-C and other infectious and inflammatory conditions. The expression levels of the genes identified were measured using RT-qPCR assays in an independent validation cohort.

Results:

Through feature selection and differential expression analysis, 11 genes with diagnostic potential were identified and taken forward into cross-platform validation using RT-qPCR. With up to 11 genes, it was possible to distinguish between MIS-C vs. KD, bacterial, and viral infections with high accuracy, with an AUC of 92.9% (95% CI: 88.2%-97.6%) in the validation cohort. The diagnostic gene signature retained its high performance when tested within the groups separately in the validation cohort: MIS-C vs. bacterial infections (AUC: 94.6%), vs. viral infections (AUC: 93.1%), and vs. KD (AUC: 89.8%).

Conclusions/Learning Points:

Despite the clinical similarities between MIS-C and other infectious and inflammatory conditions, there are key differences in gene expression profiles that can be used in diagnostic contexts. It will be necessary for the genes reported here to undergo further validation prior to their development into tests with clinical utility.

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Presenter Of 1 Presentation

Precision Medicine in the Management of Fever: The Role of Host Biomarkers

Date
Wed, 11.05.2022
Session Time
15:40 - 17:15
Session Type
Parallel Symposium
Room
DIMITRIS MITROPOULOS HALL
Lecture Time
16:07 - 16:32