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.

Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism

“…Single set of universal primers for PCR followed by sequencing for identification of bacteria has been used by other workers also [10][11][12][13][14]. In this study, we detected bacteria with only one set of PCR primers and used restriction enzyme analysis, instead of species-specific probes or sequencing, to identify bacteria.…”

“…Other studies have also shown the efficacy of broad range PCR in cases where cultures are rendered sterile by antibiotic administration [10,12,13].…”

Periodontal Inflammation: From Gingivitis to Systemic Disease?

“…22,23 When using rather insensitive bacterial culture techniques, bacteremia could be detected even in subjects with clinically healthy gingiva. The use of more sensitive molecular techniques, such as the polymerase chain reaction, 24,25 would likely prove bacterial translocation from the periodontium to be even more common than presently appreciated. While most studies of dentally related bacteremia have centered around purposeful activities such as tooth brushing, periodontal probing, and tooth extraction, it is possible that while participating in daily activities (chewing, speaking, habits, etc), minor disruptions to gingival integrity occur in a significant number of individuals with gingival inflammation.…”

The effect of human serum DNAases on the ability to detect antibiotic-killed Escherichia coli in blood by PCR

“…PCR technology has been shown to overcome some of the weaknesses of conventional culturing methods in such circumstances [2,4,5]. For instance, in a rat model of Escherichia coli bacteraemia, PCR was shown to yield a detection rate 80% higher than that of blood culture in cefotaxime-treated animals [2].…”

“…For instance, in a rat model of Escherichia coli bacteraemia, PCR was shown to yield a detection rate 80% higher than that of blood culture in cefotaxime-treated animals [2]. Nevertheless, it is still uncertain just how reliable PCR is and whether a negative result truly re¯ects the absence of bacteria in blood [4,5], as mechanisms reducing the availability of the bacterial template DNA may cause false-negative PCR results. When bacteria are killed by antibiotics, DNA may be released and then degraded by human endogenous DNAases.…”