Quantification of Bacterial DNA in Septic Intensive Care Patients in Relation to Concentration of Antibiotics and Clinical Outcome: A Prospective Observation Study

Septic syndrome caused by bloodstream infections represents a major healthcare problem worldwide and is the leading cause of mortality in the non-cardiac intensive care unit (ICU). Despite ongoing research efforts in sepsis, the mortality remains as high as 12-30%.

In severe sepsis, the time to adequate antibiotic therapy has been clearly linked to survival. However, in the emergency room, it is challenging to identify the cause behind the clinical presentation and identify patients who would benefit the most from antibiotic treatment. An indiscriminate use of antibiotics inevitably leads to bacterial resistance and unacceptable adverse events. Thus, there is a great and unmet medical need to develop novel diagnostic instruments that could inform the clinician about who would need antibiotics and who would not benefit from it. Furthermore, early identification of the causal microorganism and the inflammatory status of the patients would improve decisions about treatments and reduce administration of antibiotics.

Today the gold standard for diagnosis of bloodstream infections (BSI) is blood culture followed by identification of species and resistance pattern by standard laboratory procedures. This procedure is associated with a high specificity but also with a low sensitivity. Probable reasons for a negative blood culture despite a strong clinical suspicion could be insufficient blood volume in the culture bottles or antibiotic treatment prior to blood sampling with a subsequent failure of the bacteria to grow in the bottles.

In recent years Polymerase Chain reaction (PCR) has been proposed as a method to determine bacterial DNA based on amplification of specific subunits (16S and 18S) of bacterial ribosomal RNA. PCR require very small amount of DNA. Another advantage is the possibility to determine bacterial species from non-viable bacteria by multiplex PCRs. However most of those multiplex PCRs have shown a limited sensitivity even though they also found clinical relevant pathogens that were not detected with blood cultures. Another limitation is that multiplex-PCR only detects at maximum 95% of all sepsis causing pathogens. In a recent published study the author concluded that a tested multiplex PCR "on whole blood specimens" in adjunct to current culture-based methods provided a clinical add-on value. The future role of PCR and other molecular techniques in the clinical setting needs further evaluation.

A new method that could improve the diagnostics of sepsis and also have the possibility to follow the bacterial DNA load during antibiotic treatment is Droplet Digital PCR (ddPCR). This technique enables an absolute quantification of the sepsis causing pathogen targeting either a species specific gene or the 16S ribosomal DNA (rDNA) gene, present in several copies per bacteria. In ddPCR, the sample is divided into about 20.000 water-in-oil droplets, and in all of them the PCR-reaction occurs. Following PCR, each droplet is analyzed to determine the fraction of PCR-positive droplets giving the amount of bacterial DNA in the original sample. The absolute target DNA template concentration can then be calculated, since the size of the droplets is known, and by using Poisson distribution statistics for the correction of more than one positive reaction in the same droplet.