Identification of bacterial DNA in neutrocytic and non‐neutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction

Bruns, Tony; Sachse, Svea; Straube, Eberhard; Assefa, Sentayehu; Herrmann, Andreas; Hagel, Stefan; Lehmann, Marc; Stallmach, Andreas

doi:10.1111/j.1478-3231.2009.02073.x

Cited by 52 publications

(66 citation statements)

References 41 publications

(65 reference statements)

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“…Our 16S PCR was able to detect bacterial DNA in 100% (17/17) of culture-positive samples, and HRMA was able to correctly identify the bacterial species identified by culture in 70.6% (12/17) of positive samples in our study. The rate of positivity for bacterial DNA, 19.8% (21/106), detected from ascitic fluid samples in our study is comparable with the rates in other reported studies (3,4,8,9,11,12,(19)(20)(21)29). The overall sensitivity for detecting the presence of eubacterial DNA in an ascitic fluid sample and the specificity for the 16S PCR were high, at 100% (17/17) and 91.5% (85/89), respectively.…”

Section: Discussionsupporting

confidence: 79%

“…Although the microbiological spectrum found in our patient samples generally follows patterns previously described in other studies (3), it is interesting that 83.3% (10/12) of samples with concordant culture and HRMA results were Gram-positive organisms while other reports have Gram-negative organisms identified as the most common isolates from ascites (3,21). This higher prevalence of nosocomial infections follows the recent trend and may be due to the higher rate of exposure of our patient population to hospital environment.…”

Section: Discussionmentioning

confidence: 53%

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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

et al. 2012

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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...

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 53%

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

et al. 2012

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“…Molecular methods based on species-specific PCR or broad- range PCR for detecting the 16S rRNA gene followed by sequencing for species identification are being increasingly used for the etiological diagnosis of purulent infections, including SBP, septic arthritis, or CAPD-related peritonitis (4,7,8,12,18,19). These assays have been shown to improve the diagnostic efficiency of culture methods due to their higher sensitivities, especially with samples obtained from patients who have been exposed to antibiotics prior to specimen sampling.…”

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confidence: 99%

Performance of the LightCycler SeptiFast Test M ^grade in Detecting Microbial Pathogens in Purulent Fluids

et al. 2011

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The performance of the LightCycler SeptiFast (SF) assay was compared to that of culture methods in the detection of microorganisms in 43 purulent fluids from patients with pyogenic infections. The SF assay was more sensitive than the culture methods (86% versus 61%, respectively), irrespective of whether the infections were mono-or polymicrobial.The LightCycler SeptiFast (SF) test is a commercially available multiplex real-time PCR assay able to detect a wide range of bacterial and fungal organisms commonly involved in systemic infections (10). The SF assay has been shown to be a valuable ancillary method for the etiological diagnosis of bacteremia, sepsis, endocarditis, and more recently, periprosthetic joint infections, particularly in patients who have received antibiotics prior to diagnostic testing (1-3, 5, 6, 9, 10, 13-17). Molecular methods can allow the rapid detection of the microorganisms involved in severe pyogenic infections, thus leading to a more efficient and targeted early therapeutic intervention, which could translate into major clinical benefits for patients. To the best of our knowledge, there is no published experience on the performance characteristics of the SF assay for the detection of microorganisms in purulent fluids.A total of 43 purulent fluids obtained from 41 patients (29 male and 12 female; mean age, 64.1 years) over a period of 5 months (July to November 2010) were included in this study, as follows: 11 biliary fluid samples from patients with acute cholangitis, 7 pleural fluid samples from patients with empyema secondary to mesothelioma, lung cancer, or community-acquired and nosocomial pneumonia, 7 peritoneal fluid samples from patients with secondary peritonitis, 7 dialysis effluents from 6 patients with continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis, 5 purulent liquid samples from 4 patients with intra-abdominal abscesses secondary to abdominal surgery, 4 purulent (Ͼ250 polymorphonuclear leukocytes/l) ascites fluid samples from cirrhotic patients with spontaneous bacterial peritonitis (SBP), and 2 intra-articular fluid samples from patients with acute septic arthritis. A total of 31 of the 41 patients had been treated with broad-spectrum antibiotics prior to sampling, as follows. All patients (n ϭ 11) undergoing biliary surgery, 3 patients subjected to thoracocentesis for empyema, and 3 patients undergoing laparotomy for secondary peritonitis received perioperative antimicrobial prophylaxis with amoxicillin-clavulanic. All patients with intra-abdominal abscesses and 4 patients with secondary peritonitis were empirically treated with ciprofloxacin plus metronidazole for a median of 2 days (range, 1 to 4 days) prior to specimen sampling. Four patients with secondary empyema had been treated with imipenem, imipenem plus metronidazole, or imipenem plus vancomycin for a median of 1 day prior to specimen sampling (range, 1 to 3 days). One patient with CAPD, one patient with SBP, and one patient with septic arthritis were treated with vancomycin or ceftriaxone ...

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“…Application of culture-independent PCR-based methods for the detection of bacterial DNA (bactDNA) in ascites has been proposed as a suitable tool to improve pathogen identification in patients at risk for SBP (6) and has indicated that bactDNA derives in most cases from a single pathogen (7)(8)(9). 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).…”

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confidence: 99%

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

Krohn¹,

Böhm²,

Engelmann³

et al. 2014

J Clin Microbiol

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Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml ؊1 . Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Grampositive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples. Spontaneous bacterial peritonitis (SBP) is the most severe bacterial infection in patients with decompensated liver disease (1). As evidenced by culture-based diagnosis, Gram-negative bacteria of the genera Escherichia and Klebsiella are the most frequent cause for SBP (2, 3). However, recent data reveal that also Grampositive bacteria such as Staphylococcus and Enterococcus species may be responsible for bacterial peritonitis, especially in hospitalized patients (4, 5). Since detection rates of classical culture techniques are low in the routine setting, the causative agent remains unknown in many cases of SBP (3).Application of culture-independent PCR-based methods for the detection of bacterial DNA (bactDNA) in ascites has been proposed as a suitable tool to improve pathogen identification in patients at risk for SBP (6) and has indicated that bactDNA derives in most cases from a single pathogen (7-9). 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).Based on these findings, we analyzed a set of ascites samples using primers targeting the 16S rRNA gene for qualitative and quantitative PCR and further characterized bactDNA-positive samples by direct sequencing and terminal restriction fragment length polymorphism (T-RFLP) analysis in order to determine the frequency of detectable bactDNA in ascites from patients with end-stage liver disease and to identify the corresponding bacterial agents.A total of 356 ascites samples from 174 patients with liver cirrhosis (77.6% alcoholic, 11.5% cryptogenic, 2.9% viral hepatitis, 1.7% genetic disorders or metabolic diseases, 1.7% autoimmune hepatitis, 0.6% primary biliary cirrhosis, 3.5% nonalcoholic fatty liver disease, and 0.6% cardiac) were obtained at the University Hospital Leipzig, between February 2011 and December 2012. Thirty-seven percent of the patients (n ϭ 61) underwent several diagnostic paracenteses (mean number of paracenteses, 3.9; range, 2 to 11). All patients gave full written informed consent to the study protocol, which was approved by the local ethics committee.Ascitic fluid samples were routinely analyzed for total leukocyte count using an automated blood cell counter and for the presence of bacterial and fungal pathogens by routine microbiological culture with direct inoculation of agar plates.For culture-independe...

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Identification of bacterial DNA in neutrocytic and non‐neutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction

Abstract: Identification of ascitic bactDNA is an appropriate alternative to bacterial ascite culture for pathogen identification in patients at risk for SBP. Its prognostic relevance as a proposed marker of bacterial translocation for certain risk groups has to be further evaluated.

Cited by 52 publications

References 41 publications

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

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

Performance of the LightCycler SeptiFast Test M ^grade in Detecting Microbial Pathogens in Purulent Fluids

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

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Identification of bacterial DNA in neutrocytic and non‐neutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction

Abstract: Identification of ascitic bactDNA is an appropriate alternative to bacterial ascite culture for pathogen identification in patients at risk for SBP. Its prognostic relevance as a proposed marker of bacterial translocation for certain risk groups has to be further evaluated.

Cited by 52 publications

References 41 publications

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

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

Performance of the LightCycler SeptiFast Test M grade in Detecting Microbial Pathogens in Purulent Fluids

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

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Performance of the LightCycler SeptiFast Test M ^grade in Detecting Microbial Pathogens in Purulent Fluids