Monitoring of Patients Supported by Extracorporeal Membrane Oxygenation for Systemic Infections by Broad-Range rRNA Gene PCR Amplification and Sequence Analysis

Abstract

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...

Emerging Technologies for Molecular Diagnosis of Sepsis

“…2). Multiple studies report promising NPVs close to 97% against blood culture with the detection of multiple fastidious organisms (152,154). Similar sensitivities ranging from 37.5% to 78.6% and specificities ranging from 86.8% to 94.4% were observed in studies adjusting for clinical context by excluding contaminants (130,153).…”

“…SepsiTest can detect as few as 10 to 80 CFU/ml, with some organism bias (148,149). It has a reported sensitivity ranging from 11% to 87% and a high specificity ranging from 85% to 96% compared to blood culture in adult and pediatric patients with SIRS, sepsis, febrile neutropenia, and infectious endocarditis (130,(150)(151)(152)(153)(154) (Table 4 and Fig. 2).…”

Diagnosis of bloodstream infections from positive blood cultures and directly from blood samples: recent developments in molecular approaches

“…In a multicentre study of blood samples taken from patients with different pathologies, reported sensitivity and specificity of the test were 87% and 85.8%, respectively [54]. Later, the assay was evaluated for monitoring of patients for systemic infections during extracorporeal membrane oxygenation [57]. The assay was able to identify 45% of positive samples, 13 to 75 hours earlier than BC and detected blood culture-negative infections in 25% of patients [57].…”

“…Later, the assay was evaluated for monitoring of patients for systemic infections during extracorporeal membrane oxygenation [57]. The assay was able to identify 45% of positive samples, 13 to 75 hours earlier than BC and detected blood culture-negative infections in 25% of patients [57]. Yet, detection of any resistance genes is not available in the assay.…”

In vitro diagnosis of sepsis: a review

“…123 Conversely, the procedure is limited by its turnaround (requiring from 8 to 12 hours of work) due to several steps (DNA extraction, PCR amplification, sequencing, online sequence identification), and consequently, the risk of contaminations is increased. 124,125 In a multicenter study that evaluates the SepsiTest ® diagnostic accuracy, the assay was applied on 342 samples from 187 patients with SIRS, sepsis, or neutropenic fever; the results showed a higher rate of positivity compared to BCs. 122 As soon as its cost and time to result decrease, the assay will be considered a reliable tool for clinicians in patient care and will represent a useful supplemental method in the diagnosis of sepsis.…”