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. Coll2
1Department of Gastroenterology, CIBERehd, 2Department of Microbiology, 3Department of Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. *email@example.com
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).
- Bacterial DNA was not detected in almost half negative culture SBP episodes, questioning the bacterial etiology in these cases.
- Characteristics and clinical course in these patients differed from SBP patients with positive bacterial DNA.
- 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.