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1.
Pathogens ; 11(3)2022 Mar 11.
Article in English | MEDLINE | ID: covidwho-1760796

ABSTRACT

Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R2 range 15.1-63.4%), and per virus, with outliers up to 3 log10 reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier.

2.
J Clin Microbiol ; 60(1): e0169821, 2022 01 19.
Article in English | MEDLINE | ID: covidwho-1511413

ABSTRACT

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Laboratories , Pilot Projects
3.
Expert Rev Mol Diagn ; 21(11): 1139-1146, 2021 11.
Article in English | MEDLINE | ID: covidwho-1450340

ABSTRACT

INTRODUCTION: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown. AREAS COVERED: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective. EXPERT OPINION: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.


Subject(s)
Genome, Viral/genetics , Meningoencephalitis/diagnosis , Meningoencephalitis/virology , Metagenome/genetics , Diagnostic Tests, Routine , Humans , Metagenomics/methods
4.
J Virol Methods ; 298: 114291, 2021 12.
Article in English | MEDLINE | ID: covidwho-1433621

ABSTRACT

At the time SARS-CoV-2 was identified as the cause of coronavirus disease 2019 (COVID-19) no in vitro diagnostic (IVD) tests were available since it was a new virus. Very shortly after the release of the genomic sequence of SARS-CoV-2, laboratory-developed tests (LDTs) were developed, made available and implemented in several laboratories in the Netherlands and globally. In this study, the performance of an E-gene Sarbeco specific real-time reverse-transcriptase PCR (RT-PCR) was verified on the open modus of the geneLEAD VIII sample-to-answer platform. The results obtained from 134 clinical samples, of which 63 had been tested positive, showed almost complete concordance compared to the same PCR on the routine diagnostic systems and that was validated according to the national reference standard. The only discordant sample tested positive using the routine diagnostic workflow with a cycle threshold (CT) value of 37.7, while the sample tested negative using the geneLEAD VIII workflow. In addition, good performance was achieved in analyzing a blinded SARS-CoV-2 external quality assurance (EQA) panel. Implementation of the geneLEAD VIII platform as routine diagnostic tool resulted in testing 871 clinical samples with 115 positive results. In conclusion, the geneLEAD VIII SARS-CoV-2 workflow presented in this study showed excellent diagnostic performance and with a rapid turnaround time of approximately two hours it proved a valuable option for STAT SARS-CoV-2 testing in the absence of (rapid, CE-IVD) point-of-care testing platforms.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Testing , Humans , Point-of-Care Testing , Reference Standards , Sensitivity and Specificity
5.
Mycoses ; 64(6): 641-650, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1091024

ABSTRACT

BACKGROUND: A high prevalence of COVID-19 associated pulmonary aspergillosis (CAPA) has been reported, though histopathological evidence is frequently lacking. To assess the clinical significance of Aspergillus species in respiratory samples of mechanically ventilated COVID-19 patients, we implemented routine screening for Aspergillus in tracheal aspirate (TA). PATIENTS/METHODS: From all adult COVID-19 patients admitted to the intensive care unit (ICU), TA samples were collected twice a week for Aspergillus screening by PCR and or culture. Bronchoalveolar lavage (BAL) sampling was performed in patients with a positive screening result if possible. Clinical information was obtained from the electronic patient record and patients were categorised according to the recently published consensus case definition for CAPA. RESULTS: Our study population consisted of 63 predominantly (73%) male patients, with a median age of 62 years and total median ICU stay of 18 days. Aspergillus species were present in TA screening samples from 15 patients (24%), and probable CAPA was diagnosed in 11 (17%) patients. Triazole resistance was detected in one patient (14%). Concordance between TA and BAL was 86%, and all TA culture positives were confirmed in BAL. We were able to withhold treatment in three of fifteen patients with positive screening (20%) but negative BAL results. CONCLUSIONS: Positive culture, molecular detection and or antigen detection of Aspergillus species do not equal infection. Until we understand the clinical relevance of Aspergillus species detected in respiratory samples of COVID-19 patients, minimal-invasive screening by TA is a feasible method to monitor patients. Positive screening results should be an indication to perform a BAL to rule out upper airway colonisation.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/microbiology , Invasive Pulmonary Aspergillosis/diagnosis , Invasive Pulmonary Aspergillosis/virology , Aged , Aspergillus/genetics , Aspergillus/isolation & purification , Female , Humans , Intensive Care Units , Invasive Pulmonary Aspergillosis/drug therapy , Male , Middle Aged , Polymerase Chain Reaction/methods , SARS-CoV-2
6.
J Infect Dis ; 223(2): 206-213, 2021 02 03.
Article in English | MEDLINE | ID: covidwho-1060913

ABSTRACT

BACKGROUND: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. METHODS: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. RESULTS: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. CONCLUSIONS: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Clinical Laboratory Techniques/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Point-of-Care Testing , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Reproducibility of Results , SARS-CoV-2/genetics
7.
J Clin Virol ; 135: 104720, 2021 02.
Article in English | MEDLINE | ID: covidwho-1002741

ABSTRACT

BACKGROUND: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction. OBJECTIVE: Multicenter evaluation of the QIAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods. STUDY DESIGN: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data. RESULTS: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of CT values, showed almost complete concordance in the QIA P&A assay for samples up to CT values of 33 with one exception of CT 31. Prospectively 60 samples were tested and also showed 100 % concordance with 5 positives. The method has been automated by two centres. CONCLUSIONS: Despite an input of only 8 µL of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , Mass Screening/methods , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Clinical Laboratory Techniques/methods , Humans , Molecular Diagnostic Techniques/methods , Pandemics/prevention & control , Prospective Studies , Specimen Handling/methods , Workflow
8.
J Mol Diagn ; 22(2): 196-207, 2020 02.
Article in English | MEDLINE | ID: covidwho-832437

ABSTRACT

Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. Subsequently, the protocol was retrospectively validated using 25 clinical respiratory samples. The developed protocol using Illumina NextSeq 500 sequencing showed high repeatability. Use of the National Center for Biotechnology Information's RefSeq database as opposed to the National Center for Biotechnology Information's nucleotide database led to enhanced specificity of classification of viral pathogens. A correlation was established between read counts and PCR cycle threshold value. Sensitivity of mNGS, compared with PCR, varied up to 83%, with specificity of 94%, dependent on the cutoff for defining positive mNGS results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow, were influenza C, KI polyomavirus, cytomegalovirus, and enterovirus. Sensitivity and analytical specificity of this mNGS protocol were comparable to PCR and higher when considering off-PCR target viral pathogens. One single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses.


Subject(s)
DNA Viruses/genetics , Metagenome , Metagenomics , RNA Viruses/genetics , Respiratory Tract Infections/virology , Age Factors , Child , Computational Biology/methods , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Metagenomics/methods , Metagenomics/standards , ROC Curve , Reproducibility of Results , Respiratory Tract Infections/diagnosis , Retrospective Studies , Sensitivity and Specificity , Workflow
9.
J Clin Virol ; 132: 104632, 2020 11.
Article in English | MEDLINE | ID: covidwho-765030

ABSTRACT

BACKGROUND: Due to the emergence of the coronavirus disease 2019 (COVID-19) pandemic there is an urgent need for rapid and accurate testing on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES: The aim of this study was to assess the diagnostic performance of the GeneFinderTMCOVID-19 Plus RealAmp Kit on the ELITe InGenius sample-to-result platform, which is a commercial nucleic acid amplification test (NAT) targeting genes of SARS-CoV-2. STUDY DESIGN: Patients were eligible between March 18 and May 27, 2020, when they had respiratory symptoms that were suspected for COVID-19. The InGenius platform was compared to routine in-house NAT that was validated according to the national reference. RESULTS: Of 128 randomly selected patients, 58 (45 %) tested positive and 55 (43 %) tested negative in both platforms. Sensitivity of the InGenius platform was 100 % (95 % confidence interval 94-100). In the remaining 15 (12 %) cases E and RdRp genes were not detected in both platforms but the nucleoprotein (N) gene was tested positive by the InGenius platform. All solitary N gene positive cases were confirmed by a N-gene specific in-house validated NAT, and most of these patients could also be considered positive based on other recently available COVID-19 positive respiratory samples or highly suspected radiological findings. CONCLUSION: The InGenius platform for SARS-CoV-2 detection has excellent sensitivity, is easy to use and provides fast results. The inclusion of the N gene as a third gene target may further increase sensitivity for the diagnosis of COVID-19 in comparison to the national reference method.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics , COVID-19 Testing/methods , COVID-19 Testing/standards , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Reproducibility of Results , Sensitivity and Specificity
10.
Lancet Infect Dis ; 20(10): e251-e260, 2020 10.
Article in English | MEDLINE | ID: covidwho-693775

ABSTRACT

The term metagenomics refers to the use of sequencing methods to simultaneously identify genomic material from all organisms present in a sample, with the advantage of greater taxonomic resolution than culture or other methods. Applications include pathogen detection and discovery, species characterisation, antimicrobial resistance detection, virulence profiling, and study of the microbiome and microecological factors affecting health. However, metagenomics involves complex and multistep processes and there are important technical and methodological challenges that require careful consideration to support valid inference. We co-ordinated a multidisciplinary, international expert group to establish reporting guidelines that address specimen processing, nucleic acid extraction, sequencing platforms, bioinformatics considerations, quality assurance, limits of detection, power and sample size, confirmatory testing, causality criteria, cost, and ethical issues. The guidance recognises that metagenomics research requires pragmatism and caution in interpretation, and that this field is rapidly evolving.


Subject(s)
Metagenomics/methods , Metagenomics/statistics & numerical data , Computational Biology , Humans , Molecular Epidemiology , Research Design/standards
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