Detection of bacteraemia in patients with fever and neutropenia using 16S rRNA gene amplification by polymerase chain reaction

Abstract

Episodes of fever and neutropenia are common complications of treatment for cancer. The use of prophylactic and early empirical antibiotics has reduced mortality but decreases the sensitivity of diagnostic tests based on culture. The aim of this study was to determine the potential of a broad diagnostic approach (eubacterial) based on 16S rRNA gene amplification and sequencing to augment cultural methods of diagnosis of bacteraemia in patients with fever and neutropenia in a regional paediatric oncology centre. One hundred eleven patient-episodes of fever and neutropenia were evaluated during the study period, 17 of which were associated with positive blood cultures, as follows: Staphylococcus epidermidis (n = 6 episodes), Enterococcus faecium (n = 2), Streptococcus sanguis (n = 3), Streptococcus mitis (n = 3), Staphylococcus aureus (n = 1), Micrococcus spp. (n = 1), and Stenotrophomonas maltophilia (n = 1). Eubacterial polymerase chain reaction (PCR) detected bacterial DNA in nine of 11 blood culture-positive episodes for which a sample was available for PCR; the species identified by sequence analysis were identical to those derived from the conventional identification of the cultured isolates. Bacterial DNA was detected in 20 episodes (21 bacterial sequences) associated with negative blood cultures, 18 of which occurred in patients who were receiving antibiotics at the time of sample collection. The species presumptively identified by partial 16S rRNA gene sequencing were as follows: Pseudomonas spp. (n = 6 episodes), Acinetobacter spp. (n =5 ); Escherichia spp. (n = 3); Moraxella spp. (n = 3); Staphylococcus spp. (n = 2); Neisseria spp. (n = 1); and Bacillus spp. (n = 1). The results of this study suggest that molecular techniques can augment cultural methods in the diagnosis of bacteraemia in patients who have been treated with antibiotics.

Real-time broad-range PCR versus blood culture. A prospective pilot study in pediatric cancer patients with fever and neutropenia

“…In contrast, the blood culture method may, at least theoretically, detect bacteremia with only 1 cfu per blood culture bottle for each species growing in this system. Second, bacterial blood loads are expected to be lower after nonmyeloablative than after myeloablative chemotherapy, where sensitivity of broad-range PCR reported is higher [3,6]. In the three PCR-and blood culture-positive samples, species identification was correct in two samples, whereas the apparent discrepancy in the third sample likely resulted from the high agreement of 16S sequences of P. aeruginosa grown in culture and S. maltophilia (formerly classified as Pseudomonas maltophilia) identified by PCR.…”

“…Less than optimal sensitivity, the inherent potential for contamination, and the difficulty to distinguish background DNA from real bacteremia seem to be the main drawbacks at this moment, with blood culture remaining as the reference test. Possible optimizations are preincubation in blood culture media to enhance sensitivity, nested PCR to reduce the risk of contamination and to allow for sequencing in infection with multiple germs [3], and the simultaneous detection of DNA coding for 16S rRNA and the corresponding rRNA itself as a measure of viability [6].…”

Clinical relevance of new diagnostic methods for bloodstream infections

“…Other challenging situations that have been explored are the detection of bacteraemia in neutropenic patients with fever [16] and the detection of bacteraemia in critically ill patients [17]. However, the non-standardised nature of the assays means that it is impossible to draw any meaningful conclusions, even if the concept is appealing.…”

Copper in Immune Cells

“…They have been reported to be particularly susceptible to infection by the opportunistic pathogen Pseudomonas aeruginosa. 11 Of the Gram-positive pathogens, Staphylococcus aureus and a variety of Streptococci appear to be the most prevalent. 12 …”