Background: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic... Show moreBackground: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. Methods: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. Results: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. Conclusions: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing. Show less
Introduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the... Show moreIntroduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories.Methods: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix,One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed.Results: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection.Conclusion: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases. Show less
The main challenge of immunosuppressive therapy after solid organ transplantation is to create a new immunological balance that prevents organ rejection and does not promote opportunistic infection... Show moreThe main challenge of immunosuppressive therapy after solid organ transplantation is to create a new immunological balance that prevents organ rejection and does not promote opportunistic infection. Torque teno virus (TTV), a ubiquitous and non-pathogenic single-stranded DNA virus, has been proposed as a marker of functional immunity in immunocompromised patients. Here we investigate whether TTV loads predict the risk of common viral infection and allograft rejection in kidney transplantation recipients.In a retrospective cohort of 389 kidney transplantation recipients, individual TTV loads in were measured by qPCR in consecutive plasma samples during one year follow-up. The endpoints were allograft rejection, BK polyomavirus (BKPyV) viremia and cytomegalovirus (CMV) viremia. Repeated TTV measurements and rejection and infection survival data were analysed in a joint model.During follow-up, TTV DNA detection in the transplant recipients increased from 85 to 100%. The median viral load increased to 107 genome copies/ml within three months after transplantation. Rejection, BKPyV viremia and CMV viremia occurred in 23%, 27% and 17% of the patients, respectively. With every 10-fold TTV load-increase, the risk of rejection decreased considerably (HR: 0.74, CI 95%: 0.71-0.76), while the risk of BKPyV and CMV viremia remained the same (HR: 1.03, CI 95%: 1.03-1.04 and HR: 1.01, CI 95%: 1.01-1.01).In conclusion, TTV load kinetics predict allograft rejection in kidney transplantation recipients, but not the BKPyV and CMV infection. The potential use of TTV load levels as a guide for optimal immunosuppressive drug dosage to prevent allograft rejection deserves further validation. Show less
