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Pappas, N.

Publications and source records attributed to Pappas, N..

2 recordsLinked to original sources
bioRxiv2019

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

IntroductionExacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger for the occurrence of such exacerbations. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations.\n\nObjectivesTo study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS.\n\nStudy design88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006-2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an lllumina library preparation protocol with in-house adaptations.\n\nResultsBy mNGS, 23/88 samples tested positive. Sensitivity and specificity were both 96% for diagnostic targets (23/24 and 1067/1120, respectively). Viral pathogens only detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral species reads (p=0.008). Patients with viral pathogens had lower percentages of bacteriophages (p<0.000). No correlation was found between viral reads (species and genus level) and clinical markers.\n\nConclusionsThe mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome, and speculatively the bacterial population, during infection.

microbiology↗
bioRxiv2018

METAGENOMIC SEQUENCING FOR COMBINED DETECTION OF RNA AND DNA VIRUSES IN RESPIRATORY SAMPLES FROM PAEDIATRIC PATIENTS

IntroductionViruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables the unbiased detection of all potential pathogens in a clinical sample, including variants and even unknown pathogens. To apply mNGS in viral diagnostics, there is a need for sensitive and simultaneous detection of RNA and DNA viruses. In this study, the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections, with single tube DNA and RNA sample-pre-treatment and potential for automated pan-pathogen detection was studied.\n\nMaterials and MethodsThe sequencing protocol and bioinformatics analysis was designed and optimized including the optimal concentration of the spike-in internal controls equine arteritis virus (EAV) and phocine-herpes virus-1 (PhHV-1).The whole genome of PhHV-1 was sequenced and added to the NCBI database. Subsequently, the protocol was retrospectively validated using a selection of 25 respiratory samples with in total 29 positive and 346 negative PCR results, previously sent to the lab for routine diagnostics.\n\nResultsThe results demonstrated that our protocol using Illumina Nextseq 500 sequencing with 10 million reads showed high repeatability. The NCBI RefSeq database as opposed to the NCBI nucleotide database led to enhanced specificity of virus classification. A correlation was established between read counts and PCR cycle threshold value, demonstrating the semi-quantitative nature of viral detection by mNGS. The results as obtained by mNGS appeared condordant with PCR based diagnostics in 25 out of the 29 (86%) respiratory viruses positive by PCR and in 315 of 346 (91%) PCR-negative results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow were influenza C, KI polyomavirus, and cytomegalovirus.\n\nConclusionsSensitivity and analytical specificity of this mNGS protocol was comparable with PCR and higher when considering off-PCR target viral pathogens. All potential viral pathogens were detected in one single test, while it simultaneously obtained detailed information on detected viruses.

microbiology↗