Molecular Culture for the Diagnosis of Neonatal Sepsis: Towards Faster Recognition of Uninfected Neonates and Better Antibiotic Stewardship

Newborns often receive antibiotics for a suspicion of sepsis. Sepsis has high morbidity and mortality in newborns. Around 5% of all newborns and over 85% of very preterm born infants receive antibiotics empirically. Postnatally, antibiotics are often prescribed for presumed bacterial infections on neonatal wards, but in approximately 90-96% of these patients bacterial infection is not proven.

Rapid diagnosis of sepsis in newborns is problematic because clinical signs start subtle and are non-specific. The gold standard for diagnosis of bacterial neonatal 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 this reason, neonates 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 in the Netherlands.

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 infants, an early, quick, sensitive and specific laboratory test would be helpful to guide clinicians to decide when to discontinue antibiotics as soon as possible.

A promising technique to detect neonatal sepsis quickly and in an earlier stage compared to conventional blood culture is Molecular Culture (MC). MC is an advanced, 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 IS-pro 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 IS-pro 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-pro 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). Figure 3 shows a schematic representation of the concept of how the IS-pro procedure works.

Figure 3. Schematic representation of the concept behind the IS-pro procedure. (A) All bacterial species contain at least one IS region in their chromosome. However, many species contain multiple alleles of the IS region. These regions may vary between different alleles. Depicted here is the schematic situation in E. faecalis that contains four alleles of the IS region. Two alleles have a length of 275 nt, and the other two have a length of 377 nt. When amplified, a profile specific for E. faecalis is obtained. (B) IS profiles are highly diverse between different species. The fact that species commonly have multiple alleles with different lengths dramatically increases the differential potential between species. (C) By amplifying IS fragments with phylum-specific fluorescently labeled primers, a next layer of information is added. (D) When an IS profile is made of a sample containing multiple species from different phyla, peaks with different lengths, height, and color are found. These correspond to species, abundance, and phylum. A peak profile may be translated to a list of bacterial species by a software algorithm linked to a database of IS profiles of known bacterial species. The whole IS-pro procedure from unprocessed sample to analyzed data can be performed in 4 h.