Molecular and clinical insights into seasonal and pandemic influenza

Influenza viruses have caused significant pandemics and epidemics throughout history and continue to be a health problem in humans. New molecular diagnostic assays can be used in the clinical setting to explore relevant clinical manifestations, virus characteristics, and virus epidemiology. This thesis focuses on a variety of subjects related to the molecular diagnosis and clinical consequences of seasonal and pandemic influenza virus infections. Our findings demonstrate that molecular assays are highly sensitive and afford practical detection of resistance mutations, virulence markers and virus expression. We show that computational phylogenetics can provide accurate confirmation of an institutional influenza outbreak. Clinical observational studies reveal that immunocompromised patients can display remarkable prolonged influenza virus excretion with common antiviral resistance development, and that resistant viruses can be transmissible and pathogenic. We demonstrate that host immune responses correlate with virus-associated symptoms and sustained viral clearance. The results described in this thesis confirm that molecular diagnostic assays should be widely implemented in the clinical setting to improve influenza virus laboratory diagnostics. The inherent viral genetic variability and antigenic plasticity is a continuous incentive for new clinical and molecular research to keep up with relevant mutations and to improve our medical understanding of influenza virus infections.UBL - phd migration 201

Livestock-associated methicillin-resistant Staphylococcus aureus epidemiology, genetic diversity, and clinical characteristics in an urban region

ObjectivesWhile Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), defined as CC398, is a well-known pathogen among those working with livestock, there are indications that LA-MRSA prevalence among the general population is increasing. However, the clinical impact in urban areas remains unknown. The aim of this study was to assess the genetic epidemiology and clinical characteristics of LA-MRSA in an urban area with a limited livestock population.MethodsIn this retrospective study, we evaluated LA-MRSA strains that were collected between 2014 and 2018 from patients who received clinical care in a single urban area in Netherlands. Patient files were assessed for livestock exposure data, clinical findings, and contact tracing information. Next-generation sequencing (NGS) analysis in combination with wgMLST was conducted to assess genetic diversity and relatedness and to detect virulence and resistance genes.ResultsLA-MRSA strains were cultured from 81 patients, comprising 12% of all the MRSA strains found in seven study laboratories between 2014 and 2018. No livestock link was found in 76% of patients (n = 61), and 28% of patients (n = 23) had an infection, mostly of the skin or soft tissue. Contact tracing had been initiated in 14 cases, leading to the identification of two hospital transmissions: a cluster of 9 cases and one of 2 cases. NGS data were available for 91% (n = 75) of the patients. wgMLST confirmed the clusters detected via contact tracing (n = 2) and identified 5 additional clusters without a known epidemiological link. Relevant resistance and virulence findings included the PVL virulence gene (3 isolates) and tetracycline resistance (79 isolates).ConclusionLA-MRSA may cause a relevant burden of disease in urban areas. Surprisingly, most infections in the present study occurred in the absence of a livestock link, suggesting inter-human transmission. These findings and the presence of PVL and other immune evasive complex virulence genes warrant future surveillance and preventative measures

Overview of pH1N1 Sanger sequencing and MSCSA primers and amplicon sizes.

<p>Bp, base pairs.</p>$<p>5′ position of the first nucleotide of the forward primer in the corresponding gene;</p>#<p>5′ position of the last nucleotide of the reverse primer in the corresponding gene.</p

MSCSA and Sanger sequencing of 70 pH1N1 virus specimens.

$<p>Sanger sequencing was succesful in 63 of 65 specimens determined by MSCSA.</p>#<p>Sequence match using MSCSA and Sanger sequencing.</p>1<p>SNPs in MSCSA and not in Sanger sequencing;</p>2<p>SNPs in Sanger and not MSCSA.</p

A graphic representation of the first wave of the 2009 H1N1 pandemic in the region of Leiden (The Netherlands).

<p>A graphic representation of the first wave of the 2009 H1N1 pandemic in the region of Leiden (The Netherlands).</p

Surveillance of pH1N1 genetic markers associated with virulence or antiviral resistance.

<p>Relevant amino acid genetic positions are depicted for each corresponding gene.</p