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1.
Eur J Clin Microbiol Infect Dis ; 41(4): 663-669, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1777740

ABSTRACT

Clinical and laboratory data on newly described staphylococcal species is rare, which hampers decision-making when such pathogens are detected in clinical specimens. Here, we describe Staphylococcus massiliensis detected in three patients at a university hospital in southwest Germany. We report the discrepancy of microbiological findings between matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 16S-rRNA polymerase chain reaction, and whole-genome sequencing for all three isolates. Our findings highlight the diagnostic pitfalls pertinent to novel and non-model organisms in daily microbiological practice, in whom the correct identification is dependent on database accuracy.


Subject(s)
Blood Culture , Staphylococcus , Humans , RNA, Ribosomal, 16S/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
2.
Anal Chem ; 94(10): 4218-4226, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1721377

ABSTRACT

The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 106.7 viral copies. This value equates to 107.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Lasers , Nasopharynx , RNA, Viral/genetics , Specimen Handling/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
3.
Anal Bioanal Chem ; 413(29): 7241-7249, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1415016

ABSTRACT

Mass mapping using high-resolution mass spectrometry has been applied to identify and rapidly distinguish SARS-CoV-2 coronavirus strains across five major variants of concern. Deletions or mutations within the surface spike protein across these variants, which originated in the UK, South Africa, Brazil and India (known as the alpha, beta, gamma and delta variants respectively), lead to associated mass differences in the mass maps. Peptides of unique mass have thus been determined that can be used to identify and distinguish the variants. The same mass map profiles are also utilized to construct phylogenetic trees, without the need for protein (or gene) sequences or their alignment, in order to chart and study viral evolution. The combined strategy offers advantages over conventional PCR-based gene-based approaches exploiting the ease with which protein mass maps can be generated and the speed and sensitivity of mass spectrometric analysis.


Subject(s)
Evolution, Molecular , Mutation , SARS-CoV-2/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , COVID-19/virology , Humans , Phylogeny , SARS-CoV-2/genetics
4.
J Med Virol ; 93(9): 5481-5486, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363685

ABSTRACT

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections continue, there is a substantial need for cost-effective and large-scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS-CoV-2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT-PCR/MALDI-TOF mass spectrometry-based assay (Agena MassARRAY®) to detect SARS-CoV-2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS-CoV-2 RNA in saliva by the cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS-CoV-2 detection on the novel Agena system as well as on a conventional real-time RT-PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS-CoV-2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Diagnostic Tests, Routine/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva/virology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standards , Benchmarking , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Diagnostic Tests, Routine/instrumentation , Diagnostic Tests, Routine/methods , Humans , Limit of Detection , Nasopharynx/virology , Specimen Handling/standards , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
5.
Life Sci Alliance ; 4(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1282795

ABSTRACT

SARS-CoV-2 infection poses a global health crisis. In parallel with the ongoing world effort to identify therapeutic solutions, there is a critical need for improvement in the prognosis of COVID-19. Here, we report plasma proteome fingerprinting that predict high (hospitalized) and low-risk (outpatients) cases of COVID-19 identified by a platform that combines machine learning with matrix-assisted laser desorption ionization mass spectrometry analysis. Sample preparation, MS, and data analysis parameters were optimized to achieve an overall accuracy of 92%, sensitivity of 93%, and specificity of 92% in dataset without feature selection. We identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins, both already described as biomarkers for viral infections in the acute phase. Unbiased discrimination of high- and low-risk COVID-19 patients using a technology that is currently in clinical use may have a prompt application in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility.


Subject(s)
COVID-19/diagnosis , Machine Learning , Proteome/metabolism , Proteomics/methods , SARS-CoV-2/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Adult , Aged , Biomarkers/blood , COVID-19/epidemiology , COVID-19/virology , Female , Humans , Male , Middle Aged , Pandemics , Prognosis , Reproducibility of Results , SARS-CoV-2/physiology , Sensitivity and Specificity , Serum Amyloid A Protein/analysis
6.
Viruses ; 13(5)2021 04 22.
Article in English | MEDLINE | ID: covidwho-1202266

ABSTRACT

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.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , COVID-19/virology , Female , Genes, Viral , Genome, Viral , Humans , Male , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/isolation & purification
7.
SLAS Discov ; 26(6): 766-774, 2021 07.
Article in English | MEDLINE | ID: covidwho-1192708

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.


Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Exonucleases/antagonists & inhibitors , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
8.
Sci Rep ; 11(1): 8219, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1189285

ABSTRACT

The 2019 novel coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an unsustainable need for molecular diagnostic testing. Molecular approaches such as reverse transcription (RT) polymerase chain reaction (PCR) offers highly sensitive and specific means to detect SARS-CoV-2 RNA, however, despite it being the accepted "gold standard", molecular platforms often require a tradeoff between speed versus throughput. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) has been proposed as a potential solution for COVID-19 testing and finding a balance between analytical performance, speed, and throughput, without relying on impacted supply chains. Combined with machine learning (ML), this MALDI-TOF-MS approach could overcome logistical barriers encountered by current testing paradigms. We evaluated the analytical performance of an ML-enhanced MALDI-TOF-MS method for screening COVID-19. Residual nasal swab samples from adult volunteers were used for testing and compared against RT-PCR. Two optimized ML models were identified, exhibiting accuracy of 98.3%, positive percent agreement (PPA) of 100%, negative percent agreement (NPA) of 96%, and accuracy of 96.6%, PPA of 98.5%, and NPA of 94% respectively. Machine learning enhanced MALDI-TOF-MS for COVID-19 testing exhibited performance comparable to existing commercial SARS-CoV-2 tests.


Subject(s)
COVID-19/diagnosis , High-Throughput Screening Assays/methods , Machine Learning , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Automation , COVID-19/virology , Humans , Proof of Concept Study , SARS-CoV-2/isolation & purification
9.
Anal Chem ; 93(11): 4782-4787, 2021 03 23.
Article in English | MEDLINE | ID: covidwho-1114675

ABSTRACT

The outbreak of coronavirus disease 2019 (COVID-19) caused by SARS CoV-2 is ongoing and a serious threat to global public health. It is essential to detect the disease quickly and immediately to isolate the infected individuals. Nevertheless, the current widely used PCR and immunoassay-based methods suffer from false negative results and delays in diagnosis. Herein, a high-throughput serum peptidome profiling method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for efficient detection of COVID-19. We analyzed the serum samples from 146 COVID-19 patients and 152 control cases (including 73 non-COVID-19 patients with similar clinical symptoms, 33 tuberculosis patients, and 46 healthy individuals). After MS data processing and feature selection, eight machine learning methods were used to build classification models. A logistic regression machine learning model with 25 feature peaks achieved the highest accuracy (99%), with sensitivity of 98% and specificity of 100%, for the detection of COVID-19. This result demonstrated a great potential of the method for screening, routine surveillance, and diagnosis of COVID-19 in large populations, which is an important part of the pandemic control.


Subject(s)
COVID-19/diagnosis , Peptides/blood , SARS-CoV-2/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Area Under Curve , COVID-19/metabolism , COVID-19/virology , Case-Control Studies , Discriminant Analysis , High-Throughput Screening Assays , Humans , Least-Squares Analysis , Machine Learning , Principal Component Analysis , ROC Curve , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Tuberculosis/metabolism , Tuberculosis/pathology
10.
ACS Infect Dis ; 6(12): 3269-3276, 2020 12 11.
Article in English | MEDLINE | ID: covidwho-933654

ABSTRACT

A high resolution mass spectrometry approach has been applied for the first time to detect and characterize SARS-CoV-2 coronavirus in cell cultured and nasopharyngeal swab specimens. Peptide ions for three of the most abundant structural viral proteins (membrane, nucleocapid, and spike) are detected and assigned directly, by virtue of the high resolution and mass accuracy within the mass maps of whole virus digests, without the need for tandem mass spectrometry (MS/MS). MALDI-MS based approaches offer high sample throughput and speed, compared with those of LC-MS strategies, and detection limits at some 105 copies, or orders of magnitude less with selected ion monitoring, that compete favorably with conventional reverse transcription polymerase chain reaction (RT-PCR) strategies. The detection of signature peptides unique to SARS-CoV-2 coronavirus over those from the influenza virus allows for its unambiguous detection.


Subject(s)
COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Peptide Mapping/methods , SARS-CoV-2/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Spike Glycoprotein, Coronavirus/chemistry , Viral Matrix Proteins/chemistry , COVID-19/virology , Humans , Phosphoproteins/chemistry , Proteolysis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics
11.
J Virol Methods ; 286: 113991, 2020 12.
Article in English | MEDLINE | ID: covidwho-845838

ABSTRACT

Coronavirus disease 2019, known as COVID-19, is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The early, sensitive and specific detection of SARS-CoV-2 virus is widely recognized as the critical point in responding to the ongoing outbreak. Currently, the diagnosis is based on molecular real time RT-PCR techniques, although their implementation is being threatened due to the extraordinary demand for supplies worldwide. That is why the development of alternative and / or complementary tests becomes so relevant. Here, we exploit the potential of mass spectrometry technology combined with machine learning algorithms, for the detection of COVID-19 positive and negative protein profiles directly from nasopharyngeal swabs samples. According to the preliminary results obtained, accuracy = 67.66 %, sensitivity = 61.76 %, specificity = 71.72 %, and although these parameters still need to be improved to be used as a screening technique, mass spectrometry-based methods coupled with multivariate analysis showed that it is an interesting tool that deserves to be explored as a complementary diagnostic approach due to the low cost and fast performance. However, further steps, such as the analysis of a large number of samples, should be taken in consideration to determine the applicability of the method developed.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Nasopharynx/virology , Pneumonia, Viral/virology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Coronavirus Infections/virology , Humans , Machine Learning , Mass Screening/methods , Multivariate Analysis , Pandemics , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity
13.
Antiviral Res ; 182: 104924, 2020 10.
Article in English | MEDLINE | ID: covidwho-743859

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 µM), calpain inhibitors II and XII (IC50 ~20-25 µM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human ß-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/enzymology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Viral Nonstructural Proteins/antagonists & inhibitors , COVID-19 , Coronavirus 3C Proteases , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Glycoproteins/pharmacology , HeLa Cells , Humans , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
14.
Nat Biotechnol ; 38(10): 1168-1173, 2020 10.
Article in English | MEDLINE | ID: covidwho-690889

ABSTRACT

Detection of SARS-CoV-2 using RT-PCR and other advanced methods can achieve high accuracy. However, their application is limited in countries that lack sufficient resources to handle large-scale testing during the COVID-19 pandemic. Here, we describe a method to detect SARS-CoV-2 in nasal swabs using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and machine learning analysis. This approach uses equipment and expertise commonly found in clinical laboratories in developing countries. We obtained mass spectra from a total of 362 samples (211 SARS-CoV-2-positive and 151 negative by RT-PCR) without prior sample preparation from three different laboratories. We tested two feature selection methods and six machine learning approaches to identify the top performing analysis approaches and determine the accuracy of SARS-CoV-2 detection. The support vector machine model provided the highest accuracy (93.9%), with 7% false positives and 5% false negatives. Our results suggest that MALDI-MS and machine learning analysis can be used to reliably detect SARS-CoV-2 in nasal swab samples.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Algorithms , Biotechnology , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/statistics & numerical data , Coronavirus Infections/epidemiology , Developing Countries , False Negative Reactions , False Positive Reactions , Humans , Machine Learning , Nasal Mucosa/virology , Pandemics , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Sensitivity and Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/statistics & numerical data , Support Vector Machine
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