Longitudinal Monitoring of DNA Viral Loads in Transplant Patients Using Quantitative Metagenomic Next-Generation Sequencing.

INTRODUCTION: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. METHODS: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. RESULTS: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. CONCLUSIONS: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.

Performance of Five Metagenomic Classifiers for Virus Pathogen Detection Using Respiratory Samples from a Clinical Cohort.

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.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

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.

Viral metagenomic sequencing in the diagnosis of meningoencephalitis: a review of technical advances and diagnostic yield.

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.

Increasing diagnostic possibilities using the geneLEAD VIII platform for detection of SARS-CoV-2.

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.

Systematic screening for COVID-19 associated invasive aspergillosis in ICU patients by culture and PCR on tracheal aspirate.

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.

Rapid, Sensitive, and Specific Severe Acute Respiratory Syndrome Coronavirus 2 Detection: A Multicenter Comparison Between Standard Quantitative Reverse-Transcriptase Polymerase Chain Reaction and CRISPR-Based DETECTR.

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.

A two minute liquid based sample preparation for rapid SARS-CoV2 real-time PCR screening: A multicentre evaluation.

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.

Comparison of the GeneFinderTM COVID-19 Plus RealAmp Kit on the sample-to-result Platform ELITe InGenius to the national reference method: An added value of N gene target detection?

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.