Diagnostic utility of broad range bacterial 16S rRNA gene PCR with degradation of human and free bacterial DNA in bloodstream infection is more sensitive than an in-house developed PCR without degradation of human and free bacterial DNA.

Petra Rogina,Miha Skvarc,David Stubljar,Romina Kofol,Achim Kaasch

doi:10.1155/2014/108592

Abstract

We compared a commercial broad range 16S rRNA gene PCR assay (SepsiTest) to an in-house developed assay (IHP). We assessed whether CD64 index, a biomarker of bacterial infection, can be used to exclude patients with a low probability of systemic bacterial infection. From January to March 2010, 23 patients with suspected sepsis were enrolled. CD64 index, procalcitonin, and C-reactive protein were measured on admission. Broad range 16S rRNA gene PCR was performed from whole blood (SepsiTest) or blood plasma (IHP) and compared to blood culture results. Blood samples spiked with Staphylococcus aureus were used to assess sensitivity of the molecular assays in vitro. CD64 index was lower in patients where possible sepsis was excluded than in patients with microbiologically confirmed sepsis (P = 0.004). SepsiTest identified more relevant pathogens than blood cultures (P = 0.008); in three patients (13%) results from blood culture and SepsiTest were congruent, whereas in four cases (17.4%) relevant pathogens were detected by SepsiTest only. In vitro spiking experiments suggested equal sensitivity of SepsiTest and IHP. A diagnostic algorithm using CD64 index as a decision maker to perform SepsiTest shows improved detection of pathogens in patients with suspected blood stream infection and may enable earlier targeted antibiotic therapy.

Highlights

Sepsis affects millions of people around the globe each year and is a major cause of death [1, 2]
Spiked Blood Samples. 900 μL of ethylenediaminetetraacetic acid (EDTA) whole blood from healthy volunteers was spiked with 100 μL of serial dilutions of Staphylococcus aureus (ATCC 29213) in sterile 0.9% NaCl with final concentrations ranging from 1.5 × 10−2 colonyforming units (CFU) per mL to 1.5 × 104 CFU/mL
Staphylococcus aureus was detected in spiked blood samples by both methods: automated bacterial DNA isolation followed by an in-house developed broad range 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) assay, as well as with manual extraction using the ST protocol (Table 1)

Summary

Introduction

Sepsis affects millions of people around the globe each year and is a major cause of death [1, 2]. Blood cultures only detect viable organisms that are able to grow in the blood culture medium and often remain negative in patients that have previously received antimicrobial therapy Another criticism is that the final results from blood cultures are available too late and are of limited use for guiding initial therapy [4]. In the past few years, molecular methods of detecting microorganisms have been developed that are based on the polymerase chain reaction (PCR) In general these methods offer a shorter time to result, but they are more expensive, require highly trained staff, and have not been evaluated well in the clinical context [5]. They might be well suited to complement the standard diagnostic approach

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Conclusion