Next generation sequencing of pathogens in neonates suspected for sepsis

Neonatal sepsis, a systemic inflammatory response to an infection, is the third
leading cause of neonatal mortality and a major public health problem. It is
responsible for 13% of all neonatal mortality and 42% of deaths in the first
week of life. Recent estimates state a total of 3.0 million cases of neonatal
sepsis per year globally with a mortality of 11-19%. The diagnosis of neonatal
sepsis is difficult due to several factors: (i) especially in preterm infants,
noninfectious conditions that can resemble the symptoms of sepsis are frequent
(ii) in contrast to adult sepsis, clear-cut and harmonized sepsis criteria for
neonates are lacking or not appropriate (iii) the lack of fast and reliable
diagnostic methods, due long turnaround times as well as low sensitivity and
specificity from blood culture that represents the current standard of care,
and PCR-based assays. However, the fragility of neonatal patients in
combination with an immune system utilized with mechanisms aiming for immune
tolerance, require immediate actions. Consequently, treatment currently relies
on empiric broad-spectrum antibiotic therapy, which is in most cases not
adequately matched to the infecting pathogen or precisely timed with respect to
the duration of the infection.
This unduly antimicrobial use is itself associated with adverse clinical
outcomes due to substantial alterations of the neonatal intestinal flora,
increased risks for invasive infections by fungi or drug-resistant bacteria,
necrotizing enterocolitis (NEC) and death. New methods and approaches for an
early identification of patients suffering from infection together with the
identification of the causative microbes are therefore urgently needed to
reduce the incidence and mortality of neonatal sepsis.
It is recently shown, that circulating cell-free DNA (cfDNA) from blood plasma
of adult septic patients not only is a suitable but a more sensitive and
specific analyte to identify pathogens by next-generation sequencing and a
bioinformatics workflow coupled with statistic tests to reveal a clinical
relevance. NGS-based diagnosis offers many advantages: (i) it is an open
platform, providing the opportunity to detect bacterial, fungal and viral
pathogens in a single assay, (ii) it is quantitative by counting sequence reads
and calculating statistical significances and can therefore potentially
discriminate between unspecific colonization/contamination or infection (iii)
it provides unbiased and untargeted sequence information on any DNA in patient
specimens offering higher sensitivity and specificity.
The aim of this research approach is thus to provide an alternative technology
for a timely and untargeted identification of pathogens causing sepsis.
Furthermore, a high negative predictive value of the pathogen identification
test coupled with fast results on the host*s immune status could lead to
reduced antibiotic consumption in this patient collective.
In previous studies, the general applicability and superior reliability of NGS
diagnostics of underlying pathogens from cfDNA over blood culture in adult
septic patients could be shown. This promising methodology would be of
tremendous value particularly for neonates as being one of the two groups at
highest risk at the extremes of age with peak incidence and mortality.
Preliminary results to adapt the technology from adult patients cover
successful cfDNA isolation and NGS diagnostics from limited plasma volumes to
match the considerably smaller blood volumes in neonates. A pilot study with
neonates suspected for sepsis is now mandatory to identify pathogens with this
technique. Therefore, for example, the algorithms for pathogen identification
need to be adapted and trained to detect the differences in bacterial burden
and species composition from neonatal plasma samples.

Observational pilot study to investigate NGS as a reliable method to identify
sepsis in neonates. NGS is proposed as a method to improve clinical
identification, diagnosis and stratification of neonatal sepsis and provide
basis for a more adequate and targeted therapy for neonates.

This pilot study is an investigators-initiated study to indicate proof of
concept of next generation sequencing of plasma cell-free DNA for the diagnosis
of sepsis in neonates. Patients will be enrolled in a monocentre study design
at the neonatal department in the Maxima Medical Centre Veldhoven in the
Netherlands. Inclusion time will be estimated for 5 months.
NGS is a recently developed technique which will be tested in this pilot study
in a new clinical setting. Therefore, 10 patients clinically suspected for
sepsis and 10 non-infected patients are enrolled in the study. A blood culture
is obtained in infants who are suspected for sepsis and an additional 0.5 ul
blood will be obtained for the study. In the non-infected control group in
addition to a clinical indication for blood sampling, 0.5 ml blood is obtained.

Risks associated with participation are considered negligible and the burden is
considered minimal since a research subject is not exposed to an extra puncture
besides the clinical indicated puncture to obtain the blood culture. It
concerns 0.5 ml extra blood which is nearby 1% of the total blood volume of a
neonate born after 26 weeks of gestation with a birth weight of 800 grams. Most
infants in the study will be older and have a higher birth weight.