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.
Blood cultures are still gold standard, but are generally assumed to have a low sensitivity
for the diagnosis of sepsis in newborns and children and are time consuming. Cases of sepsis
may be missed by cultures and a more sensitive diagnostic test such as molecular tests as the
MC may be useful. Advances in microbial technology have led to the development of rapid
molecular methods such as MC, that may be more sensitive than culture. Multiple novel
molecular techniques, such as quantitative PCR, broad range conventional PCR and multiplex
PCR, have been studied to detect neonatal sepsis. However, these techniques are directed at
specific species which makes it impossible to detect all bacterial species. Thus, anything
that is not explicitly searched for will be missed. In contrast, MC has the ability to detect
every bacterial species that can cause bacterial neonatal sepsis.
The MC technique is validated to detect these pathogenic bacteria in bodily fluids. A series
of papers validating all aspects of the MC technique have been published last years.
Furthermore, MC is being used in hospitals on the intensive care unit to detect bacteria in
otherwise sterile specimens like blood, but also on samples obtained from abscesses. A proof
of principle study on 39 neonates, suspicious for EOS, of whom additional blood samples were
taken from both umbilical cords as well as from peripheral phlebotomy, showed that MC was
able to detect a pathogenic bacterial strain that was highly likely the causative organism
for sepsis in one infant that was clinically ill. Conventional culture did not yield any
results for this patient. 2 additional MC samples showed strains that were likely to be
contaminants in infants that were clinically well-appearing, were conventional culture
remained negative. No other discrepancies were seen. This study also showed that MC on blood
samples that were spiked with prevalent bacterial strains for neonatal sepsis, showed a very
high agreement with quantitative PCR as a control diagnostic.Larger studies are needed to
corroborate diagnostic accuracy given the very low incidence of culture proven sepsis.
Results from another study using the MC technique to detect bacteria in human body fluid are
promising. In this study 66 samples were collected and tested by conventional culture and MC.
In 100% of samples with a positive culture, the MC was also positive. In five samples, the
conventional culture turned out to be negative, whereas the MC was positive. The case
histories of these five patients were obtained and suggested that the MC findings were highly
clinically relevant, and thus may have higher sensitivity compared to conventional blood
culture.
In summary, the quick detection makes MC potentially better equipped to guide clinical
decision making for management of sepsis in newborns and children. However, the suitability
of MC in this specific population has not been investigated adequately. Therefore, a high
quality study should be performed to determine the diagnostic accuracy of the MC for sepsis
in neonates and children and whether this technique may replace the conventional blood
culture.
