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Molecular diagnostic test applied to ascitic fluid
SIRS-Lab GmbH
Winzerlaer Straße 2
D-07745 Jena
Introduction
Spontaneous bacterial peritonitis (SBP) is a common and life-threatening complication of patients with ascites and cirrhosis of the liver. The mortality accounts for up to 30-50% and such patients die within several hours after infection. An early diagnosis and subsequent antibiotic therapy are prerequisites for the survival of the patients. The clinical standard for a diagnosis of SBP is the increased number of polymorphic neutrophilic cells in the ascites (> 250/μl), independent of the results obtained with ascites cultures1,2. An increased cell count is not detectable in all patients. Positive ascites cultures are typically found in 40-90% of all SBP cases3,4. A specific detection of bacterial and fungal pathogens associated with SBP, which is relevant for further specific antimicrobial therapies, could be established on the use of culture-based methods, comparable to the blood culture technique. The latter being the standard method in clinical microbiology laboratories to demonstrate the presence of pathogens in patients suspected of systemic infections. However, this technique exhibits some drawbacks due to the patient’s antibiotic treatment prior to sample withdrawal, a low abundance of causative agents, non-cultivable organisms, and the period of usually 2 to 4 days to obtain results. This time-toresult of blood cultures is mostly too long to initiate an effective therapy. Nucleic acid amplification methods (e. g. PCR) allow a more rapid target detection compared to culture-based methods. However, due to the small reaction volumes of those methods, the sensitivities are not yet competitive. VYOO™ as the combination of culture-independent pathogen-derived nucleic acids concentration and PCR-based species detection was proven to be a fast and sensitive method with high specificity and sensitivity for pathogen analysis.
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Fig. 1 Survey of current pathogen detection methods. |
Concentration of bacterial and fungal nucleic acids
The LOOXSTERŽ protocol was optimized for a concentration of bacterial and fungal DNA from whole blood samples. However, the underlying LOOXSTERŽ method is intended for a source independent sample DNA concentration, e. g. from ascites. Ascites samples exhibit varying human cell counts and therefore dissimilar eukaryotic DNA backgrounds. However, the prokaryotic/eukaryotic DNA ratios were proven to be minute.
LOOXSTERŽ was developed for the specific concentration of bacterial and fungal DNA. The system is based on SIRS-Lab’s proprietary PUREPROVEŽ technology that specifically binds bacterial and fungal DNA and removes human background nucleic acids. After a cell lysis protocol, total DNA is prepared and applied to a column for the specific concentration of the minute quantity of bacterial and fungal DNA from a mixture containing a huge human DNA background. The following concentration step is based on DNA affinity chromatography using a matrix-immobilized DNA binding protein which recognizes definite motives within the bacterial and fungal DNA. When a complex DNA mixture is applied onto the matrix, more than 90% of the human background DNA is removed. The final bacterial + fungal/human DNA ratio is significantly higher in favor of the first, which substantially increases the sensitivity of downstream PCR protocols and simultaneously reduces the time-to-result.
For further information on the PUREPROVEŽ technology, please refer to the LOOXSTERŽ technical and application notes.
Downstream pathogen detection
Subsequently, the concentrated prokaryotic and fungal nucleic acids can be analyzed by PCR. The multiplex PCR test VYOO™ was developed for sepsis causative bacterial and fungal species. Relevant primers were selected and optimized to provide a basis for a directive antibiotic therapy. The results are visualized with gelelectrophoresis or hybridization techniques. Isolation, concentration, and detection methods of the bacterial and fungal DNA are combined within the VYOO™ detection system.
Data on the presence of distinct bacterial species are available within less than one working day which is not possible with culture techniques (e. g. ascites or blood culture; 2-4 days).
Objective of the study
The focus of the study was to determine whether VYOO™ was suitable, fast, and sensitive enough to detect bacterial infections of the ascitic fluid of patients who are suspected of spontaneous bacterial peritonitis. The results have been compared to standard cell count methods and additional ascites cultures. For quantifying the efficiency of the DNA concentration step, qPCR using adjusted primers was applied. These primers recognize regions within the conserved 16S-rDNA coding sequence. VYOO™ Multiplex PCR was used to identify the definite causative agents. The procedure was designed to additionally supply valuable information with respect to the initiation of an antimicrobial therapy.
Results
The data obtained by VYOO™ were compared to the gold standard results (increased number of polymorphic neutrophilic cells in the ascites ≥ 250/μl) and those of ascites cultures. The efficiency of the LOOXSTERŽ procedure according to the DNA content that was applied to the column was determined using 16S-rDNA PCR before and after LOOXSTERŽ (see Figure 1).
Although LOOXSTERŽ was optimized for higher total DNA amounts which are to be expected in whole blood samples, a significant concentration of bacterial DNA was achieved from ascitic fluids despite varying amounts of total DNA.
Focusing on 19 samples of the abovementioned subgroup of 14 patients, VYOO™ was positive in all cases where ascites cultures were positive and increased neutrophilic cell numbers were counted. Additionally, one patient was proven to be multi-infected by E. faecalis, E. coli, and E. faecium, where the ascites culture and the total cell number (< 250 cells/μl) were both negative. Table and Figure 1 summarize the results of the study and Table 2 gives a selection of case reports of patients where SBP was confirmed by the procedures applied in the study.
The NAT-methods applied yielded positive results in all three selected cases. The total cell numbers and neutrophilic cell counts have been increased and the ascites cultures were positive in two cases, respectively. Additionally, VYOO™ detected a multi-infection in one case (patient 3) in which neither the cell counts nor the copy number (as determined by qPCR) were increased. The patient was initially medicated with Ceftriaxon/Metronidazol. Three days after withdrawal of blood and ascitic fluid, the blood culture was positive for E. faecalis whereas the parallel ascites culture remained negative. Thus, the therapy was changed to Tazobac, which does not inhibit growth of E. faecium. The latter, E. coli, and E. faecalis were later found in a blood culture and wound swabs. However, the same three organisms (E. coli, E. faecalis, and E. faecium) were also found by VYOO™ on the first day. This demonstrates that VYOO™ delivers the same results within hours whereas blood or ascites cultures require several days. The use of VYOO™ allows rapid pathogen detection and therefore an initiation of a pathogen directed, appropriate, and early antibiotic therapy.
Based on these results it has to be stated that neither the current gold standard method nor a 16S-rDNA-qPCR or ascites culture alone are sufficient for SBP diagnosis in this case, besides the fact that the first two methods gain no information on specific antibiotic treatment and the latter requires several days. Figure 2 gives an example for the identification of E. coli in ascitic fluid by VYOO™ Multiplex PCR.
Conclusions
The results of the study are to be summarized as follows:
1. The LOOXSTERŽ protocol as part of VYOO™ is easily applicable for ascites samples and increases the sensitivity of subsequent NAT methods.
2. The combination of LOOXSTERŽ and nucleic acids amplification-based methods in the VYOO™ test system are useful to provide therapeutic relevant results within less than one day.
3. VYOO™ led to a significant increase in sensitivity, specificity, and reduction of time compared to the gold standard and culture-based methods.
Acknowledgments
Samples were provided by the Clinic of Internal Medicine, Department for Gastroenterology, Hepatology and Infectiology, and experiments were performed at the Institute of Medical Microbiology, Friedrich-Schiller-University Jena, Germany.
References
1 Scemama-Clergue et al. Gut 26:332-335, 1985
2 Rimola et al. J Hepatol 32:142-153, 2000
3 Runyon et al. Gastroenterology 95:1351-1355, 1988
4 Siersema et al. J Clin Microbiol 30:667-669, 1992
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