Direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information.

BACKGROUND: Acute respiratory tract infections (RTI) cause substantial morbidity during childhood, and are responsible for the majority of pediatric infectious diseases. Although most acute RTI are thought to be of viral origin, viral etiology is still unknown in a significant number of cases. OBJECTIVES: Multiplexed whole genome sequencing (WGS) was used for virome determination directly on clinical samples as proof of principle for the use of deep sequencing techniques in clinical diagnosis of viral infections. STUDY DESIGN: WGS was performed with nucleic acids from sputum and nasopharyngeal aspirates from four pediatric patients with known respiratory tract infections (two patients with human rhinovirus, one patient with human metapneumovirus and one patient with respiratory syncytial virus), and from four pediatric patients with PCR-negative RTI, and two control samples. RESULTS: Viral infections detected by routine molecular diagnostic methods were confirmed by WGS; in addition, typing information of the different viruses was generated. In three out of four samples from pediatric patients with PCR-negative respiratory tract infections and the two control samples, no causative viral pathogens could be detected. In one sample from a patient with PCR-negative RTI, rhinovirus type-C was detected. Almost complete viral genomes could be assembled and in all cases virus species could be determined. CONCLUSIONS: Our study shows that, in a single run, viral pathogens can be detected and characterized, providing information for clinical assessment and epidemiological studies. We conclude that WGS is a powerful tool in clinical virology that delivers comprehensive information on the viral content of clinical samples.

Transcriptome kinetics of circulating neutrophils during human experimental endotoxemia.

Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1ß. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.