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1.
Diagnostics (Basel) ; 13(8)2023 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-37189571

RESUMO

We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.

2.
Diagnostics (Basel) ; 12(10)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36292212

RESUMO

The prefusion spike protein of SARS-CoV-2 binds advanced glycation end product (AGE)-glycated human serum albumin (HSA) and a higher mass (hyperglycosylated/glycated) immunoglobulin (Ig) G3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress syndrome (ARDS) as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct serum dilution, and disulphide bond reduction, method was developed and applied to plasma samples from SARS-CoV-2 seronegative (n = 30) and seropositive (n = 31) healthcare workers (HCWs) and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for the development of COVID-19 ARDS as a result of SARS-CoV2 infection. Furthermore, rapid direct analysis of serum/plasma samples by MALDI-ToF for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions.

3.
Viruses ; 14(9)2022 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-36146765

RESUMO

Applying MALDI-ToF mass spectrometry as a clinical diagnostic test for viruses is different from that of bacteria, fungi and other micro-organisms. This is because the systems biology of viral infections, the size and chemical nature of specific viral proteins and the mass spectrometry biophysics of how they are quantitated are fundamentally different. The analytical challenges to overcome when developing a clinical MALDI-ToF mass spectrometry tests for a virus, particularly human pathogenic enveloped viruses, are sample enrichment, virus envelope disruption, optimal matrix formulation, optimal MALDI ToF MS performance and optimal spectral data processing/bioinformatics. Primarily, the instrument operating settings have to be optimized to match the nature of the viral specific proteins, which are not compatible with setting established when testing for bacterial and many other micro-organisms. The capacity to be a viral infection clinical diagnostic instrument often stretches current mass spectrometers to their operational design limits. Finally, all the associated procedures, from sample collection to data analytics, for the technique have to meet the legal and operational requirement for often high-throughput clinical testing. Given the newness of the technology, clinical MALDI ToF mass spectrometry does not fit in with standard criteria applied by regulatory authorities whereby numeric outputs are compared directly to similar technology tests that have already been authorized for use. Thus, CLIA laboratory developed test (LDT) criteria have to be applied. This article details our experience of developing a SAR-CoV-2 MALDI-ToF MS test suitable for asymptomatic carrier infection population screening.


Assuntos
COVID-19 , Viroses , Vírus , Bactérias/química , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas Virais , Viroses/diagnóstico
4.
Int J Mol Sci ; 23(11)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35682724

RESUMO

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype's differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Antivirais , Humanos , Imunoglobulina G , Nucleocapsídeo , SARS-CoV-2
5.
Diagnostics (Basel) ; 10(10)2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987950

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. The need to rapidly screen vast numbers of a country's population in order to control the spread of the infection is paramount. However, the logistical requirement for reagent supply (and associated cost) of RT-PCR based testing (the current front-line test) have been hugely problematic. Mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. Here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bio- and physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high-throughput and reproducible rapid process. Utilizing MALDI-ToF mass spectrometric detection to viral envelope glycoproteins in a systems biology-multidisciplinary team approach, we have achieved a multifaceted clinical MALDI ToF MS screening test, primarily (but not limited to) SARS-CoV-2, with direct application to other future epidemics/pandemics that may arise. The clinical information generated not only includes SARS-CoV-2 coronavirus detection-(Spike protein fragments S1, S2b, S2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total Ig light chain peak) and a measure of the viral immune response (elevated intensity of IgA heavy chain peak). The advantages of the method include; (1) ease of sampling, (2) speed of analysis, and much reduced cost of testing. These features reveal the diagnostic utility of MALDI-ToF mass spectrometry as a powerful and economically attractive global solution.

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