Two new commercially available universal rRNA gene PCR plus sequencing tests, SepsiTest and universal microbe detection (UMD; Molzym, Bremen, Germany), were evaluated using blood specimens and heart valves from 30 patients with suspected infectious endocarditis (IE). The sensitivity of PCR (85%) was nearly twice as high as that of culture (45%), which in 10/20 IE cases presumably stayed negative as a consequence of growth inhibition of the pathogens by antibiotics. Further, PCR provided the basis for reclassification of 5/10 non-IE cases into IE cases. Culture-negative infections were identified by PCR, including single infections due to streptococci and Gram-negative bacteria (Escherichia coli, Haemophilus parainfluenzae) and mixed infections involving two Gram-positive bacteria or Candida spp. with Gram-positive bacteria. The new commercial tests proved to be of value for the rapid diagnosis of IE, particularly in cases of culture-negative infections. Issues regarding the feasibility of these tests for routine use are discussed.
Extracorporeal membrane oxygenation (ECMO) represents a temporary life-saving therapy for respiratory or circulatory failure, but infections during ECMO support are a life-threatening complication. Surface-related infections of ECMO are mentioned, but rarely described in the literature. A universal rDNA polymerase chain reaction (PCR) test was used to investigate the potential microbiological colonization of membrane oxygenators (MOs) in 20 patients undergoing ECMO. The overall patient-based positivity by PCR was 45%. Gram-positive bacteria (71%) represented the most abundant microorganisms on MO surfaces, followed by Gram-negative bacteria (22%) and fungi (7%). The most frequently detected causative pathogens were staphylococci (58%). Bacterial mixed infections represented 56% of all infections. In four PCR-positive cases, the pathogens detected on the MO surfaces were also found by blood culture or by culture of specimens obtained from the infectious focus. In conclusion, hollow fiber membranes of MOs can be colonized by microorganisms and appear to be a potential source of bacterial and fungal infections in ECMO patients. These infections may pose an increased risk for clinical worsening. As a consequence, persistent septic complications have to be discussed as an indication for MO exchange. The initial results suggest that the applied PCR assay is a valuable tool to investigate MOs.
The PCR results demonstrate that the molecular test is a useful diagnostic tool for the rapid diagnosis of IE. Furthermore, the molecular diagnosis had a significant, direct impact on the therapy of IE. This suggests that using PCR can improve antibiotic treatment, particularly in cases of culture-negative IE. Consequently, molecular analysis of micro-organisms in HV samples should be performed routinely where preoperative diagnosis remains unclear.
eThe rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative. P atients supported by extracorporeal membrane oxygenation (ECMO) are at high risk for microbial systemic infections (1, 2) and therefore are continuously monitored by clinical and inflammatory markers. Microbiological results are used to tailor antibiotic treatment to the most effective regimen. Unfortunately, etiologies are typically identified within 1 to 2 days, and up to 31% of cultures remain negative because of limited blood volume analyzed, growth failure of fastidious organisms, inhibition of growth of pathogens by antibiotics, and other unknown reasons (3, 4). Molecular methods are discussed as tools for improvement of the diagnosis of septicemia (5, 6). Among them, panbacterial and panfungal rRNA gene PCR followed by sequencing identifies the broadest range of pathogens (7-9). Here, we evaluated the usefulness of a broad-range PCR test, SepsiTest (6, 10-14), for the monitoring of patients for DNA of pathogens in the blood during ECMO.The study was approved by the Ethics Committee of Hannover Medical High School. SepsiTest results were not considered for the administration of antibiotics. ECMO criteria included cardiac or respiratory failure with beginning or preexisting organ failure.Single pairs of aerobic/anaerobic Bactec Plus bottles (BD, Heidelberg, Germany) and Bactec Mycosis-IC/F bottles were incubated each with 10 ml of blood collected from a venipuncture. Incubation was for 7 (bacteria) or 14 (fungi) days in case of negative results. Species were identified using Vitek 2 (bioMérieux, Marcy l'Etoile, France). Other samples were analyzed according to standard microbiological operations of the laboratory.Duplicate samples (1 ml) of EDTA blood (from the same venipuncture as blood culture [BC]) were analyzed using SepsiTest (Molzym, Bremen, Germany) (6, 10-14), which supplies protocols and reagents for DNA extraction, 16S and 18S rRNA gene PCR, and negative, positive, and internal PCR controls. Amplicon sequencing was done by an overnight service. BLAST results (www.sepsitest-blast.net) were classified at the species (Ն99% sequence identity) and genus (Ն97%) levels (12). Corynebacteria, viridans streptococci, coagulase-negative staphylococci (CNS), and Klebsiella oxytoca/Enterobacter cloacae were not resolvable at the species level. Mixed sequences were resolved by Ripseq analysis (Isentio, Paradis, Norway).Defined criteria were applied to interpret BC-negative, SepsiTest-positive results. "Probable" systemic infection was supposed if (i) results were supported by cultured material (12) before, during, or after ECMO and/or (ii) organisms were found in repeated blood draws within 48 h that were regarded clinically significant on the grounds of the c...
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