In this study, we successfully employed ISH to detect pathogens in the blood from patients with decompensated LC in whom blood culture results were negative for bacterial infection. These findings suggested that bacterial translocation cannot be always detected by conventional blood culture because SBP is known to occur after bacterial translocation, defined as the passage of bacteria from the intestine or colon through the intestinal epithelial cells and entrapment in the mesenteric lymph nodes [8, 11]. After bacterial translocation, bacteria are thought to enter the systemic bloodstream and access ascitic fluid, which exhibits low bactericidal capacity [9, 12,13,14]. Bacterial translocation has been demonstrated in some studies in both human and animal models of LC [15, 16]. However, it is unclear how SBP develops from bacterial translocation because this event cannot be detected easily by conventional blood culture [11]. Such et al. reported that bacterial DNA can be detected simultaneously in blood and ascitic fluid [17, 18], using a polymerase chain reaction (PCR)-based method. Although this method may provide evidence of the relationship between bacterial translocation and SBP, no studies have compared PCR-based methods with blood culture. Therefore, we hypothesized that ISH could be applied to decompensated LC patients to detect bacteria and may be helpful for selecting patients who may have an infection earlier and determining the proper antibiotic to use if bacteria are present in the systemic bloodstream after bacterial translocation.
ISH was first developed to enable early diagnosis of sepsis within 1 day, using ISH of the bacterial genomes existing in neutrophils after phagocytosis. This method was reported to be four times as sensitive as blood culture for detection of bacteria in patients with sepsis [7]. The other advantages of the ISH method are that it can eliminate potential contamination and is not affected by antibiotic use, as it analyzes pathogens already captured into neutrophils by phagocytosis. Thus, the probability of false-positive results is low with this procedure. Enomoto et al. developed a new probe mixture, designated a global bacteria (GB) probe, which was capable of detecting all relevant bacterial strains. Using ISH, this probe showed positive results in 10 of 11 SBP cases and negative results in none of 40 non-SBP cases in ascites [6]. Bacteria causing SBP are frequently gram-negative rods, such as Escherichia coli and K. pneumoniae, or can be Streptococcus species [10]. Such et al. studied bacterial DNA to show that Escherichia coli were the most frequently identified bacteria [18]. This is consistent with our results demonstrating that Enterobacteria were frequently detected. Therefore, even five specific probes without the global bacteria probe may be useful for detection of bacteria in patients with decompensated LC. These results suggest that early detection of specific bacteria causing SBP and early therapeutic intervention using appropriate antibiotics are additional advantages of ISH for patients with LC and SBP. However, further studies are needed to confirm this assumption. Additionally, blood cultures beyond ISH tests are thought to be necessary for such patients because bacteria other than the five species probed by ISH may be detected and because drug susceptibility tests cannot be performed by ISH.
Interestingly, in our study, some clinical parameters were related to the results of ISH tests, including the presence of fever and Child-Pugh scores. These results may also be related to the occurrence of bacterial translocation. Cirera et al. detected enteric organisms increasingly from mesenteric lymph nodes in patients with or without cirrhosis according to the Child-Pugh score: 3.4% in A, 8.1% in B, and 30.8% in C [15]. Bacteria in neutrophils were detected in 47% of patients without fever and in 54% of patients whose CRP levels were below 1 mg/dL in our study. Surprisingly, bacterial translocation may already be present in asymptomatic patients with LC having ascites. Evans et al. reported that 3.5% of all outpatients with cirrhosis had SBP, and 1.9% of these patients had bacterascites [19]. Moreover, several studies have also demonstrated that more severe liver failure is associated with lower CRP levels [20, 21]. Administration of antibiotics may be considered to prevent further deterioration of sepsis or SBP in decompensated LC patients positive for ISH; these patients have no clinical symptoms at this point.
Recent reports have shown that serum albumin functions to maintain oncotic pressure and has immunomodulatory and antioxidant effects. Albumin infusions were found to reduce the incidence of renal failure and mortality in patients with SBP [22, 23]. Patients with positive results by ISH had higher Child-Pugh scores and showed a tendency of lower serum albumin levels (Table 4). If hypoalbuminemia indicates immunological deterioration in patients with decompensated LC, patients who are positive for ISH may be required to receive albumin infusions early in addition to antibiotics.
The limitations of this study include the small number of subjects. Obviously, a larger controlled study will be needed to validate the results and confirm the usefulness of ISH.