Poster Presentations

Session Title: Category 2b. CIRRHOSIS AND ITS COMPLICATIONS: b. CLINICAL ASPECTS
Presentation Date: Apr 15, 2010

BACTERIAL DNA DETECTION BY REAL-TIME PCR AND 16S RDNA GENE SEQUENCING IN SPONTANEOUS BACTERIAL PERITONITIS

G. Soriano¹*, O. Esparcia², M. Montemayor², C. Guarner-Argente¹, R. Pericas², X. Torras¹, N. Calvo³, E. Román¹, F. Navarro², C. Guarner¹, P. Coll^2
1Department of Gastroenterology, CIBERehd, ²Department of Microbiology, ³Department of Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. *gsoriano@santpau.cat

Background: Despite of inoculation into blood culture bottles, ascitic fluid culture is negative in 50% of cases of spontaneous bacterial peritonitis (SBP). AIM. To determine whether 16S rDNA gene detection by real-time PCR and sequencing increases the efficacy of culture in microbiological diagnosis of SBP.
Patients and methods: We included all cirrhotic patients with SBP (ascitic fluid neutrophil count ≥250/mm³, n=55): culture was positive in 25 (SBP+C) and negative in 30 (SBP-C). We also included a group of cirrhotic patients with sterile ascites (SA, ascitic fluid neutrophil count < 250/mm³ and negative culture, n=20), and another group of patients with neoplasic ascites (NA, n=27). Bedside inoculation into blood culture bottles and real-time PCR and sequencing of 16S rDNA gene were performed in ascitic fluid.
Results: Bacterial DNA was detected in 23/25 (92%) cases from SBP+C group, 16/30 (53%) in SBP-C (p=0.004 respect to SBP+C), 12/20 (60%) in SA (p=0.01 respect to SBP+C and pNS respect to SBP-C) and 0/27 in NA (p< 0.001 respect to other groups). Sequencing identified 12 cases from SBP+C group to genus or species level (8 agreed with culture, 4 did not), 6 in SBP-C and 6 in SA. In the remaining cases with positive PCR, sequencing did not yield a definitive bacterial identification. Analyzing all SBP episodes (n=55) and comparing patients with positive (n=39) and negative bacterial DNA (n=16), the former showed worse liver function (Child-Pugh AB/C 19/20 vs 13/3, p=0.03), a trend to a higher inflammatory response (blood leukocyte count 11880±9235 vs 6588±2713/mm³, p=0.002; SIRS 48.7% vs 18.7%, p=0.06; ascitic fluid neutrophil count 7214±12854 vs 2649±4183/mm³, pNS) and a trend to higher in-hospital mortality (25.6% vs 6.2%, pNS) and 3-month probability of mortality (31% vs 6%, p=0.058).
Conclusions:

1. Bacterial DNA was not detected in almost half negative culture SBP episodes, questioning the bacterial etiology in these cases.
2. Characteristics and clinical course in these patients differed from SBP patients with positive bacterial DNA.
3. Methodology used did not always identify amplified bacterial DNA; this could be due to presence of polymicrobial DNA as a consequence of previous polymicrobial translocations and require further studies.