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Pathogen detection with VYOO(TM)
SIRS-Lab GmbH
Winzerlaer Straße 2
D-07745 Jena
Sepsis is the most common cause of death in hospitalized patients worldwide. Inadequate antibiotic therapy represents an independent determinant of patients’ mortality.
Promptness of pathogen detection and the appliance of appropriate antibiotics are the cornerstones of management of severe bacterial infections. Furthermore, resistance to antibiotics is an increasing public health problem that can be attributed largely to their overuse. Thus, rapid diagnostic tests with both superior positive and negative predictive values over current techniques are sorely needed.
The specific detection of sepsis-causing organisms from whole blood still suffers from unsatisfactory clinical utility. Currently, blood culture is the gold standard method for pathogen detection in patients suspected of systemic infections. This technique is known to have many drawbacks, especially with regard to antibiotic pre-treatment, low abundance, and non-cultivable organisms. It usually takes 3 to 5 days to obtain a result from blood culture, which is too late to initiate an effective therapy. Amplification-based methods, such as PCR, deliver results faster. However, their sensitivity is lower than that of culture-based methods. A sensitivity increase of PCR methods can be achieved by specific concentration of DNA.
VYOO™ compensates the mentioned drawbacks with the included sample preparation system LOOXSTER®. The system is based on SIRS-Lab’s proprietary PUREPROVE® technology, which specifically concentrates bacterial and fungal DNA and removes human background DNA. Therewith, LOOXSTER® increases the sensitivity of downstream culture-independent detection methods. With VYOO™, this principle is included in a system for pathogen detection.
VYOO™ detects ~40 bacterial and fungal species that cause life-threatening infections. The definition of those species is based on blood culture study results. Further, important antibiotic resistances are identified in the same process. The multiplex PCR analysis is designed for the examination of the data by gelelectrophoresis or hybridization methodologies. VYOO™ delivers results of high therapeutic value within ~6 hours.
The LOOXSTER® effect
Concentration of bacterial and fungal DNA
Whole blood samples of sepsis patients contain complex DNA mixtures with a high human DNA background causing a very low bacterial plus fungal/human DNA ratio (1:2 x109; see Figure 1). At first, a total cell lysis protocol is performed. The following concentration step uses the PUREPROVE® technology incorporated in LOOXSTER®. PUREPROVE® is based on a protein which recognizes definite motives within the bacterial and fungal DNA. A PUREPROVE®-based matrix removes more than 90% of the human background DNA. The final bacterial plus 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 PUREPROVE® technology included in the LOOXSTER® system, please consult the LOOXSTER® technical note.
The VYOO™ principle
Fast and precise results for guided antibiotic therapy
The multiplex PCR protocol is designed for the highly specific amplification of a series of bacterial and fungal targets. Information about the presence of bacterial and fungal species is available within ~6 hours. This accelerates pathogen detection by 2 to 4 days. The analysis of the generated amplicons is processed by gradient polyacrylamide gelelectrophoresis or hybridization techniques.
Table 1 gives an overview on the bacterial and fungal species/genera and resistances which can be determined with VYOO™. The multiplex primers were selected and optimized to provide a basis for further therapeutic steps, for instance a targeted antibiotic therapy. The primer selection is based on comprehensive clinical studies on sepsis (see Figure 1). All primers were tested against many different pathogens which can be present in blood samples but are irrelevant for sepsis.
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Figure 1: Frequencies of sepsis causative pathogens as determined in 11 clinical studies. Individual species were designated only in a part of the studies, which accounts for their percental distribution as species and within genera/families and groups. The boxplot data were calculated using R (R Development Core Team, 2007). *without S. pneumoniae
Bodmann und Vogel, Chemotherapie Journal (2001) 10:43-56; Cockerill et al., Clin Inf Dis (1997) 24:403-418; Focht und Adam, Klinikarzt (2004) 33:35-38; Fridkin and Gaynes, Clinics in Chest Medicine (1999) 20:303-316; Geffers et al., Anästhesiol Intensivmed Notfallmed Schmerzther (2004) 39:15-19; Jones et al., Ann Clin Microbiol Antimicrobials (2004) 3:1-11; Karlowsky et al., Ann Clin Microbiol Antimicrobials (2004) 3:1-8; Kübler et al., Med Sci Monit (2004) 10:635-641; Styers et al., Ann Clin Microbiol Antimicrobials (2006) 5:1-9; Geerdes et al., Clin Inf Dis (1992) 15:991-1102; Vincent et al., Crit Care Med (2006) 34:344-353; R: A language and environment for statistical computing. R Foundation for Statistical Computing (2007) Vienna, Austria, ISBN 3-900051-07-0, URL http:// www.R-project.org
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1primer-pool I; 2primer-pool II; *tested species were detected by the Staphylococcus spp. primer (primer-pool II); #tested species were detected by the Prevotella spp. primer (primer-pool II); §tested species were detected by the Fungi spp. primer |
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Figure 2: Selection of amplicons generated by VYOO™ Multiplex PCR. M 1: VYOO™ Marker; 1: Morganella morganii (232 bp), 2: Streptococcus agalactiae (302 bp), 3: Enterobacter cloacae (364 bp), 4: Enterococcus faecalis (531 bp), 5: Streptococcus pyogenes (662/737 bp), 6: Streptococcus dysgalactiae (737 bp), 7: Streptococcus bovis (861 bp), 8: Streptococcus sanguinis (925 bp), M 2: 100 bp DNA-Ladder (Roth, Karlsruhe, Germany) |
Pathogen detection with high therapeutic value
VYOO™ enables the timely appliance of appropriate antibiotics and addresses the current drawbacks in sepsis pathogen detection. The combination of sensitivity and promptness generates increased therapeutic value. VYOO™ carries a significant potential to guide antibiotic therapy in the early therapeutic window that determines the course and prognosis of the septic patient. Moreover, its high utility helps to identify those patients in whom antibiotic therapy can safely be deescalated.
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