Identification of Bacterial Pathogens in Ascitic Fluids from Patients with Suspected Spontaneous Bacterial Peritonitis by Use of Broad-Range PCR (16S PCR) Coupled with High-Resolution Melt Analysis

Abstract

bSpontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBP rely on culture techniques for proper diagnosis, although recent reports suggest that the presence of bacterial DNA in peritoneal fluid in patients with cirrhosis and ascites is an indicator of SBP. A previously published broad-range PCR (16S PCR) coupled with high-resolution melt analysis (HRMA) was compared with standard culture techniques for diagnosis of SBP in 106 peritoneal fluid samples from patients with suspected SBP. The sensitivity and specificity for 16S PCR for detecting eubacterial DNA compared with those of standard culture techniques were 100% (17/17) and 91.5% (85/89), respectively. Overall, HRMA concordance with species identification was 70.6% (12/17), although the 5 samples that were discordant at the species level were SBP resulting from a polymicrobial infection, and specieslevel identification for polymicrobial infections is outside the capability of HRMA. Both the broad-range 16S PCR and HRMA analysis provide useful diagnostic adjunctive assays for clinicians in detecting and identifying pathogens responsible for SBP. Spontaneous bacterial peritonitis (SBP) is a common and potentially fatal bacterial infection in patients with cirrhosis and ascites, occurring in 10 to 30% of patients, with in-hospital mortality rates ranging from 20 to 30% (1, 2, 6-8, 12). It is secondary to impaired humoral and cellular immune responses that result in indirect intestinal bacterial translocation into the ascitic fluid (1, 2, 6-8, 16, 20). SBP is also associated with a poor long-term prognosis for patients, as mortality rates can reach 50 to 70% at 1 year (2). Given that timely and appropriate antibiotic treatment can improve the clinical outcome, rapid and accurate diagnostic methods for early detection of eubacterial infection responsible for SBP and identification of the causative organisms involved could be particularly useful in acute care settings. Recent attention drawn to the changing microbial and resistance patterns attributed to the increasing use of antibiotic prophylaxis and invasive procedures in such patients further underscores the importance of identifying the causative pathogen to ensure adequate antibiotic coverage (13).Current laboratory diagnosis of SBP is defined as Ն250 polymorphonuclear (PMN) cells/ml and a positive culture from ascitic fluid from the patient (1-13, 15-17, 19-22, 29). Unfortunately, the prolonged turnaround time (1 to 2 days) of culture limits its utility for directing antibiotic selection in acute care settings. Ascitic fluid culture has also been reported to be negative in approximately 20% of patients with clinical manifestations suggestive of SBP and an ascitic PMN count of Ͼ250, so-called culture-negative neutrocytic ascites. On the other hand, a low ascitic PMN count (Ͻ250) with positive culture can also occur in another SBP variant called bacterascites, or monom...

Application of Qualitative and Quantitative Real-Time PCR, Direct Sequencing, and Terminal Restriction Fragment Length Polymorphism Analysis for Detection and Identification of Polymicrobial 16S rRNA Genes in Ascites

“…However, recent studies using 16S rRNA-based fingerprinting analyses have shown that ascites may also be polymicrobial (10,11) and that the bacterial spectrum is broader than that previously reported in context with SBP (11).…”

The prognostic significance of bacterial DNA in patients with decompensated cirrhosis and suspected infection

“…Different methods result in a broad variability in detected AF bactDNA ranging from around 10% (18,27,28) with single-plex or multiplex PCR, 30-40% with 16s rRNA PCR (7,13,14,16,21), up to 60% with realtime TaqMan PCR (26), which is well comparable to the prevalence of AF bactDNA in our study cohort. Although the results obtained by some techniques support the traditional concept of monomicrobial BT present in serum and AF (7,13,14,16,21), more recent studies using 16S rRNA-based fingerprinting analyses rather suggest polymicrobial peritoneal diversity questioning this canonical concept (29)(30)(31)(32).…”

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection

“…Prior to PCR analysis, all samples were centrifuged at 8000 rpm for 10 minutes to separate DNA from intact bacterial cells. The supernatant was used for reference standard PCR analysis using a previously described 16 S real-time PCR assay (22–27). Briefly, each PCR reaction was performed in a total volume of 50 µL, utilizing 30 µL of PCR master mix and 20 µL of sample.…”