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Exacerbations 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. 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. To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS. 88 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 Illumina library preparation protocol with in-house adaptations. By mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p<0.001). No correlation was found between viral reads and clinical markers. The 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 pathogenic respiratory viral 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 during infection, with potential consequences for the bacterial populations, during infection. [ABSTRACT FROM AUTHOR]

Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation. [ABSTRACT FROM AUTHOR]

The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting. [ABSTRACT FROM AUTHOR]