Children's Health Assessment and Molecular Pathogen Identification for Optimized Novel Sepsis Therapy

Newborns and children often receive antibiotics for a suspicion of sepsis. Sepsis has high morbidity and mortality in newborns and children. Up to 50% of children in the Netherlands are prescribed at least one course of antibiotics in the first 4 years of life. Postnatally, antibiotics are often prescribed for presumed bacterial infections on neonatal and pediatric wards, but in approximately 30% of these patients bacterial infection is not proven.

Rapid diagnosis of sepsis in newborns and children is problematic because clinical signs start subtle and are non-specific. The gold standard for diagnosis of bacterial sepsis is a conventional blood culture. Unfortunately, bacterial culture is time-consuming (time to result up to 36-72 hours) and lacks sensitivity in this population for sepsis. For this reason, children and infants with risk factors for infection or clinical signs and symptoms of infection are treated with antibiotics empirically at initial sepsis suspicion, awaiting results of the conventional blood culture. Currently more than 85% of very preterm born infants (gestational age <30 weeks) receive antibiotics for the risk of early-onset sepsis (EOS) in the Netherlands and approximately two-third are evaluated at least once for late-onset sepsis (LOS) . Also older children are often prescribed antibiotics empirically for presumed sepsis, awaiting results of the blood culture.

There is increasing evidence that, apart from antibiotic resistance, the use of antibiotics in the neonatal period and during childhood alters the microbiome with an increased risk of immediate and long-term adverse effects, such as increased risk for asthma, obesity, allergies and inflammatory bowel diseases (IBD). To avoid unnecessary treatment of non-infected children, an early, quick, sensitive and specific laboratory test would be helpful to guide clinicians to decide when to discontinue antibiotics as soon as possible.

Another technique to detect neonatal sepsis quickly and in an earlier stage compared to conventional blood culture is Molecular Culture (MC). MC is a rapid molecular based culturing technique that is able to identify bacteria within 4 hours after blood sampling. In short, MC is a DNA-based profiling technique, differentiating between bacterial species based on species-specific differences in 16S-23S rDNA interspacer (IS) region nucleotide length by using phylum-specific fluorescently labelled polymerase chain reaction (PCR) primers. A standard MC procedure consists of two separate PCRs. In the first PCR two different primers are added, one primer fluorescently labeling members of the phyla Firmicutes, Actinobacter, Fusobacteria and Verrucomicrobia (FAFV), whereas the second primer labels members of the Bacteroidetes phylum. In the second PCR, a third labeled primer is added targeting members of the phylum Proteobacteria. Subsequently, these PCR products can be amplified and DNA fragments can be separated based on their nucleotide length. Eventually, a typical MC microbial profile will be created, consisting of a set of color-labeled peaks. Each peak representing an individual bacterial operational taxonomic unit (OTU) depending on the nucleotide length of the IS fragment, length of these peaks demonstrating the concentration of this particular OTU, whereas peak colors provides information about the present phyla (FAFV, Bacteroidetes or Proteobacteria).

In bacterial sepsis, a bacterium has reached the otherwise sterile bloodstream causing sepsis as a result of dysregulation of the host immune response and/or a reaction of bacterial endotoxins. Both blood culture and MC can detect bacteria and give a positive result (in case a bacterium has been cultured) or a negative result (in case no bacteria was detected). In case the tests are positive, both techniques also show which bacteria was found, which could be used to change antibiotic regimen to target that specific cultured bacterium. However, the process of MC can be finished within 4 hours, which is much shorter than the incubation period of the gold standard blood culture which is 36-72 hours. When there is no sepsis (and thus no bacteria in the bloodstream), the MC will turn out negative also within 4 hours and thus may guide clinicians to stop antibiotics in uninfected children much faster compared to the conventional blood culture.