Discussion

Sepsis is the tenth most common cause of death in the US [1]. Blood culture and other microbiological cultures represent the standard methods the clinician has to confirm the presence of the pathogen responsible for the septic picture and to start adequate antimicrobial and other adjuvant therapies. Unfortunately the failure rate of these tests remains high. There are valid reasons why bacterial strains that cause clinical sepsis are not recovered by routine blood culture. For example, prior antibiotic treatment may render them nonviable. A second reason is that the organisms may be genuinely hard to grow or require special conditions for growth. Some sources of sepsis may produce transient bacteremia. Organisms may be sequestered in tissue foci, in resident macrophage, in the capillary bed with few being released in the circulation. Under such circumstances, the identification of bacterial DNA in blood and hemofilter by 16S rRNA gene analysis may facilitate bacterial DNA recovery from the clinically septic patients with negative blood cultures. Recent studies have shown that the automated blood culture systems failed to detect symptomatic bacteremia in critically ill patients with a reported incidence of 3-6% [16-18]. and of these, Pseudomonas aeruginosa is the most common organism causing false-negative cultures [16, 17].

ARF requiring RRT is a common finding during severe sepsis in the ICU. These patients are particularly prone to severe infections and still little is known about the possibility of an extracorporeal clearance of pathogenic agents [19].

The aim of our study was to evaluate the feasibility of 16S rRNA gene analysis in the systemic blood, filter and UF of critically ill septic patients treated by CRRT. In the present study, in a cohort of 9 critically ill patients with a diagnosis of severe sepsis and ARF, the technique described showed satisfactory results in terms of the presence or absence of the microorganism, even when standard blood culture was negative. Despite continued evidence of septic shock, repeated blood cultures of only 2 patients (22%) were positive. However, bacterial DNA was identified from the blood of 6 patients (67%), as well as the 2 septic patients with documented positive blood cultures. Interestingly, in all patients bacterial DNA was found on the filter blood side, even when the DNA test in the blood was negative. These findings suggest that he-mofilters might work as concentrators: synthetic membranes used during RRT at the ultrafiltration rates constantly adsorb proteins within their hollow fibers. The thickness of this protein layer at the blood-membrane interface progressively increases, resulting in a potential deposit of bacteria or bacterial DNA beneath this protein gel. In case of an undetectable amount of bacteria and bacterial DNA from blood, this amount may be detected within the 'concentrating' hemofilter. Finally, bacterial DNA was detected in the dialysate compartment of the hemofilters in 78% of subjects. Bacterial DNA weighs several billion Daltons [20] and thus it does not traverse a standard RRT filter with a cutoff of 50 kD. Fragmented bacterial DNA, whose size is less than the hemofilter pores, might cross the membrane and be present in the dialysate compartment. Also, these bacterial DNA fragments must include the 540-bp conserved region of the bacterial 16S rRNA gene detected by the primers for the PCR amplification. In light of these results, a potential role of hemofilters might be speculated in clearing the bacterial circulating DNA by adsorption from the bloodstream. The clinical impact of this clearance was not evaluated in the present study. However, bacterial DNA was not found in the centrifuged and precipitated UF. A recent study by Hansard et al. [21] demonstrated that pathogenic bacteria can be recovered by the culture of UF in clinically septic, blood culture-negative patients. On the contrary, in our patients we could not recover bacterial DNA in the UF of CVVH using DNA detection with the molecular technique. This was confirmed even in septic patients with positive blood culture (subjects 2 and 3A). The explanations for the incongruity between the detection of bacterial DNA in the UF and dialysate compartment may be: (1) bacterial DNA in the UF is highly diluted and undetectable even by centrifugation or precipitation, and (2) hemofilters have adsorptive properties to bacteria/bacterial DNA on both sides of the membrane.

This technology has a number of limitations. First, this method needs personnel experienced in molecular biology and must be performed under careful quality control to prevent false-positive results. Sequencing analysis is more expensive than the traditional identification methods and in most cases is not useful because of the co-presence of more then one microorganism. A different strategy is imposed if there is a situation in which mixed culture is likely, such as in critically ill patients admitted to the ICU for a long duration where superinfection is often found. In this case, it is impossible to interpret and compare the sequencing results with a standard database and the results will be reported as 'unsequentiable'. The 16S rRNA amplification can however be used as a rapid and very sensitive screening test of positive versus negative samples, while specific primers for defined bacteria must be used to further identify the suspected pathogenic organisms. Furthermore, this method detects bacterial DNA or the fragmentation of bacterial DNA, which may remain in the body despite the absence of viable microorganisms (as demonstrated in subject 3B). Therefore, the diagnostic result of the test should be interpreted simultaneously with the clinical presentation of the patient.

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